CA3196886A1 - Polypeptides for use in the treatment and prevention of neurodegenerative diseases - Google Patents
Polypeptides for use in the treatment and prevention of neurodegenerative diseasesInfo
- Publication number
- CA3196886A1 CA3196886A1 CA3196886A CA3196886A CA3196886A1 CA 3196886 A1 CA3196886 A1 CA 3196886A1 CA 3196886 A CA3196886 A CA 3196886A CA 3196886 A CA3196886 A CA 3196886A CA 3196886 A1 CA3196886 A1 CA 3196886A1
- Authority
- CA
- Canada
- Prior art keywords
- derivative
- isolated polypeptide
- functional fragment
- salt
- amino acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 208000015122 neurodegenerative disease Diseases 0.000 title claims abstract description 64
- 230000004770 neurodegeneration Effects 0.000 title claims abstract description 61
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 264
- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 202
- 229920001184 polypeptide Polymers 0.000 title claims description 198
- 238000011282 treatment Methods 0.000 title description 27
- 230000002265 prevention Effects 0.000 title description 5
- 238000000034 method Methods 0.000 claims abstract description 107
- 208000010877 cognitive disease Diseases 0.000 claims abstract description 64
- 208000024891 symptom Diseases 0.000 claims abstract description 56
- 230000004153 glucose metabolism Effects 0.000 claims abstract description 46
- 230000006999 cognitive decline Effects 0.000 claims abstract description 42
- 230000013016 learning Effects 0.000 claims abstract description 38
- 230000006870 function Effects 0.000 claims abstract description 37
- 230000003920 cognitive function Effects 0.000 claims abstract description 34
- 230000006377 glucose transport Effects 0.000 claims abstract description 32
- 230000007787 long-term memory Effects 0.000 claims abstract description 31
- 230000006403 short-term memory Effects 0.000 claims abstract description 27
- 239000012634 fragment Substances 0.000 claims description 86
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 83
- 150000003839 salts Chemical class 0.000 claims description 75
- 208000024827 Alzheimer disease Diseases 0.000 claims description 50
- 108090000623 proteins and genes Proteins 0.000 claims description 43
- 239000003814 drug Substances 0.000 claims description 41
- 230000014509 gene expression Effects 0.000 claims description 41
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 32
- 230000036542 oxidative stress Effects 0.000 claims description 26
- 230000001771 impaired effect Effects 0.000 claims description 25
- 208000018737 Parkinson disease Diseases 0.000 claims description 23
- 206010012289 Dementia Diseases 0.000 claims description 22
- 229940124597 therapeutic agent Drugs 0.000 claims description 22
- 210000002569 neuron Anatomy 0.000 claims description 21
- 239000008194 pharmaceutical composition Substances 0.000 claims description 21
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 230000001965 increasing effect Effects 0.000 claims description 20
- 210000004556 brain Anatomy 0.000 claims description 19
- 208000027061 mild cognitive impairment Diseases 0.000 claims description 19
- 108010021188 Superoxide Dismutase-1 Proteins 0.000 claims description 18
- 102100038836 Superoxide dismutase [Cu-Zn] Human genes 0.000 claims description 18
- 239000003085 diluting agent Substances 0.000 claims description 18
- 239000003937 drug carrier Substances 0.000 claims description 17
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 17
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 claims description 16
- 206010027175 memory impairment Diseases 0.000 claims description 16
- 208000035475 disorder Diseases 0.000 claims description 15
- 108091006296 SLC2A1 Proteins 0.000 claims description 14
- 208000026072 Motor neurone disease Diseases 0.000 claims description 13
- 208000005264 motor neuron disease Diseases 0.000 claims description 13
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 claims description 12
- 230000008499 blood brain barrier function Effects 0.000 claims description 12
- 210000001218 blood-brain barrier Anatomy 0.000 claims description 12
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 claims description 12
- 208000019901 Anxiety disease Diseases 0.000 claims description 11
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 claims description 11
- 102000014461 Ataxins Human genes 0.000 claims description 10
- 108010078286 Ataxins Proteins 0.000 claims description 10
- 206010008025 Cerebellar ataxia Diseases 0.000 claims description 10
- 208000009415 Spinocerebellar Ataxias Diseases 0.000 claims description 10
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 claims description 10
- 201000004562 autosomal dominant cerebellar ataxia Diseases 0.000 claims description 10
- 208000002320 spinal muscular atrophy Diseases 0.000 claims description 10
- 108091052347 Glucose transporter family Proteins 0.000 claims description 9
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 9
- 108091000117 Tyrosine 3-Monooxygenase Proteins 0.000 claims description 9
- 102000048218 Tyrosine 3-monooxygenases Human genes 0.000 claims description 9
- 238000002648 combination therapy Methods 0.000 claims description 9
- 201000011240 Frontotemporal dementia Diseases 0.000 claims description 8
- 230000036506 anxiety Effects 0.000 claims description 8
- 239000000544 cholinesterase inhibitor Substances 0.000 claims description 8
- 229930195712 glutamate Natural products 0.000 claims description 8
- 230000006872 improvement Effects 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 208000023105 Huntington disease Diseases 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 230000009467 reduction Effects 0.000 claims description 7
- 208000020925 Bipolar disease Diseases 0.000 claims description 5
- 208000024412 Friedreich ataxia Diseases 0.000 claims description 5
- 208000009829 Lewy Body Disease Diseases 0.000 claims description 5
- 201000002832 Lewy body dementia Diseases 0.000 claims description 5
- 208000024777 Prion disease Diseases 0.000 claims description 5
- 229940122041 Cholinesterase inhibitor Drugs 0.000 claims description 4
- 208000019736 Cranial nerve disease Diseases 0.000 claims description 4
- 102000018711 Facilitative Glucose Transport Proteins Human genes 0.000 claims description 4
- 229940123730 Orexin receptor antagonist Drugs 0.000 claims description 4
- 206010039966 Senile dementia Diseases 0.000 claims description 4
- 229950008995 aducanumab Drugs 0.000 claims description 4
- 201000006417 multiple sclerosis Diseases 0.000 claims description 4
- 206010044652 trigeminal neuralgia Diseases 0.000 claims description 4
- 208000018127 Idiopathic intracranial hypertension Diseases 0.000 claims description 3
- 208000001381 pseudotumor cerebri Diseases 0.000 claims description 3
- 230000007774 longterm Effects 0.000 claims description 2
- 230000032258 transport Effects 0.000 claims description 2
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 claims 3
- 206010020772 Hypertension Diseases 0.000 claims 1
- 239000008280 blood Substances 0.000 claims 1
- 210000004369 blood Anatomy 0.000 claims 1
- 239000008279 sol Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 10
- 210000004027 cell Anatomy 0.000 description 99
- 235000001014 amino acid Nutrition 0.000 description 25
- 125000000539 amino acid group Chemical group 0.000 description 25
- 229940024606 amino acid Drugs 0.000 description 24
- 150000001413 amino acids Chemical class 0.000 description 24
- 230000000694 effects Effects 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 21
- 201000010099 disease Diseases 0.000 description 17
- 229940079593 drug Drugs 0.000 description 17
- FNQVICDIQNFNHD-NOQIVBDNSA-N CC(C)C[C@@H](NC(=O)[C@H](NC(=O)[C@@H](CO)NC(=O)[C@H]1CCCN1C(=O)[C@@H](CC(C)C)NC(=O)[C@@H](Cc1ccccc1)NC(=O)[C@@H](Cc1ccccc1)NC(=O)[C@H](NC(=O)[C@@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](N)Cc1c[nH]c2ccccc12)[C@H](C)O)[C@H](C)O)C(=O)N[C@H](Cc1c[nH]c2ccccc12)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCNC(N)=N)C(=O)N[C@H](CCCCN)C(O)=O Chemical compound CC(C)C[C@@H](NC(=O)[C@H](NC(=O)[C@@H](CO)NC(=O)[C@H]1CCCN1C(=O)[C@@H](CC(C)C)NC(=O)[C@@H](Cc1ccccc1)NC(=O)[C@@H](Cc1ccccc1)NC(=O)[C@H](NC(=O)[C@@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](N)Cc1c[nH]c2ccccc12)[C@H](C)O)[C@H](C)O)C(=O)N[C@H](Cc1c[nH]c2ccccc12)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCNC(N)=N)C(=O)N[C@H](CCCCN)C(O)=O FNQVICDIQNFNHD-NOQIVBDNSA-N 0.000 description 16
- 150000008575 L-amino acids Chemical class 0.000 description 16
- 102000012944 human tumor-cells apoptosis factor Human genes 0.000 description 16
- 108010065114 human tumor-cells apoptosis factor Proteins 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 15
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 13
- 229930195725 Mannitol Natural products 0.000 description 13
- 239000000594 mannitol Substances 0.000 description 13
- 235000010355 mannitol Nutrition 0.000 description 13
- 230000015654 memory Effects 0.000 description 12
- 230000001681 protective effect Effects 0.000 description 11
- 102100037597 Brain-derived neurotrophic factor Human genes 0.000 description 10
- 150000008574 D-amino acids Chemical class 0.000 description 10
- 102000008165 X-Box Binding Protein 1 Human genes 0.000 description 9
- 108010035430 X-Box Binding Protein 1 Proteins 0.000 description 9
- 206010028980 Neoplasm Diseases 0.000 description 8
- 230000009286 beneficial effect Effects 0.000 description 8
- 230000007423 decrease Effects 0.000 description 8
- 238000011161 development Methods 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 8
- 238000002483 medication Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 201000011510 cancer Diseases 0.000 description 7
- 230000006378 damage Effects 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 230000001225 therapeutic effect Effects 0.000 description 7
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 6
- 208000000044 Amnesia Diseases 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 206010029260 Neuroblastoma Diseases 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- 239000006180 TBST buffer Substances 0.000 description 6
- 102000004142 Trypsin Human genes 0.000 description 6
- 108090000631 Trypsin Proteins 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 230000001149 cognitive effect Effects 0.000 description 6
- 238000003745 diagnosis Methods 0.000 description 6
- 239000012588 trypsin Substances 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 102000042092 Glucose transporter family Human genes 0.000 description 5
- 230000006735 deficit Effects 0.000 description 5
- 230000008449 language Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 241000282412 Homo Species 0.000 description 4
- 208000026139 Memory disease Diseases 0.000 description 4
- 208000009668 Neurobehavioral Manifestations Diseases 0.000 description 4
- 206010044565 Tremor Diseases 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 230000000996 additive effect Effects 0.000 description 4
- 230000006399 behavior Effects 0.000 description 4
- 230000003925 brain function Effects 0.000 description 4
- -1 carrier Substances 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 239000002612 dispersion medium Substances 0.000 description 4
- ADEBPBSSDYVVLD-UHFFFAOYSA-N donepezil Chemical compound O=C1C=2C=C(OC)C(OC)=CC=2CC1CC(CC1)CCN1CC1=CC=CC=C1 ADEBPBSSDYVVLD-UHFFFAOYSA-N 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 230000033001 locomotion Effects 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000006984 memory degeneration Effects 0.000 description 4
- 208000023060 memory loss Diseases 0.000 description 4
- 230000007903 penetration ability Effects 0.000 description 4
- 235000020183 skimmed milk Nutrition 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 206010061818 Disease progression Diseases 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 3
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 3
- WGAZVKFCPHXZLO-SZMVWBNQSA-N Leu-Trp-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N WGAZVKFCPHXZLO-SZMVWBNQSA-N 0.000 description 3
- 108010025020 Nerve Growth Factor Proteins 0.000 description 3
- AXIOGMQCDYVTNY-ACRUOGEOSA-N Phe-Phe-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 AXIOGMQCDYVTNY-ACRUOGEOSA-N 0.000 description 3
- KWMZPPWYBVZIER-XGEHTFHBSA-N Pro-Ser-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWMZPPWYBVZIER-XGEHTFHBSA-N 0.000 description 3
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 3
- 102000040945 Transcription factor Human genes 0.000 description 3
- 108091023040 Transcription factor Proteins 0.000 description 3
- GQYPNFIFJRNDPY-ONUFPDRFSA-N Trp-Trp-Thr Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CC=3C4=CC=CC=C4NC=3)C(=O)N[C@@H]([C@H](O)C)C(O)=O)=CNC2=C1 GQYPNFIFJRNDPY-ONUFPDRFSA-N 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 230000032683 aging Effects 0.000 description 3
- 108010062796 arginyllysine Proteins 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 210000004958 brain cell Anatomy 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 238000011088 calibration curve Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 230000005750 disease progression Effects 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- ASUTZQLVASHGKV-JDFRZJQESA-N galanthamine Chemical compound O1C(=C23)C(OC)=CC=C2CN(C)CC[C@]23[C@@H]1C[C@@H](O)C=C2 ASUTZQLVASHGKV-JDFRZJQESA-N 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 108010057821 leucylproline Proteins 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- BUGYDGFZZOZRHP-UHFFFAOYSA-N memantine Chemical compound C1C(C2)CC3(C)CC1(C)CC2(N)C3 BUGYDGFZZOZRHP-UHFFFAOYSA-N 0.000 description 3
- 230000003340 mental effect Effects 0.000 description 3
- 230000036651 mood Effects 0.000 description 3
- 230000016273 neuron death Effects 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 230000003449 preventive effect Effects 0.000 description 3
- 230000000750 progressive effect Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- 101100230254 Drosophila melanogaster Glut3 gene Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102000007072 Nerve Growth Factors Human genes 0.000 description 2
- 208000012902 Nervous system disease Diseases 0.000 description 2
- 208000027626 Neurocognitive disease Diseases 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- XSVMFMHYUFZWBK-NSHDSACASA-N Rivastigmine Chemical compound CCN(C)C(=O)OC1=CC=CC([C@H](C)N(C)C)=C1 XSVMFMHYUFZWBK-NSHDSACASA-N 0.000 description 2
- 101150052594 SLC2A3 gene Proteins 0.000 description 2
- 206010041250 Social phobia Diseases 0.000 description 2
- 102000019197 Superoxide Dismutase Human genes 0.000 description 2
- 108010012715 Superoxide dismutase Proteins 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 2
- 229960003942 amphotericin b Drugs 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 229940121375 antifungal agent Drugs 0.000 description 2
- 239000003429 antifungal agent Substances 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 102000052586 bactericidal permeability increasing protein Human genes 0.000 description 2
- 230000003542 behavioural effect Effects 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 210000004081 cilia Anatomy 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000002784 cytotoxicity assay Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 230000006866 deterioration Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000010874 in vitro model Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000000266 injurious effect Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 229960004640 memantine Drugs 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 230000000626 neurodegenerative effect Effects 0.000 description 2
- 230000004031 neuronal differentiation Effects 0.000 description 2
- 230000003955 neuronal function Effects 0.000 description 2
- 230000000324 neuroprotective effect Effects 0.000 description 2
- 230000004792 oxidative damage Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 210000001428 peripheral nervous system Anatomy 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920005862 polyol Polymers 0.000 description 2
- 150000003077 polyols Chemical class 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 208000020016 psychiatric disease Diseases 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 239000008158 vegetable oil Substances 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 206010001497 Agitation Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 206010003591 Ataxia Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 208000006096 Attention Deficit Disorder with Hyperactivity Diseases 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- 101150035467 BDNF gene Proteins 0.000 description 1
- 101150043508 BPI gene Proteins 0.000 description 1
- 206010006100 Bradykinesia Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 208000028698 Cognitive impairment Diseases 0.000 description 1
- 206010010947 Coordination abnormal Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 206010012239 Delusion Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 208000027534 Emotional disease Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 206010017577 Gait disturbance Diseases 0.000 description 1
- 208000011688 Generalised anxiety disease Diseases 0.000 description 1
- 102000058062 Glucose Transporter Type 3 Human genes 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 208000004547 Hallucinations Diseases 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 208000006083 Hypokinesia Diseases 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 1
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 208000005870 Lafora disease Diseases 0.000 description 1
- 208000014161 Lafora myoclonic epilepsy Diseases 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 102000015336 Nerve Growth Factor Human genes 0.000 description 1
- 208000011644 Neurologic Gait disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 102000002512 Orexin Human genes 0.000 description 1
- 206010033664 Panic attack Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 206010034719 Personality change Diseases 0.000 description 1
- 206010034912 Phobia Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 241000425347 Phyla <beetle> Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 108091006298 SLC2A3 Proteins 0.000 description 1
- 208000000810 Separation Anxiety Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010034546 Serratia marcescens nuclease Proteins 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 229950010081 abrineurin Drugs 0.000 description 1
- 108010034788 abrineurin Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000006241 alcohol protecting group Chemical group 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 150000001408 amides Chemical group 0.000 description 1
- 125000006242 amine protecting group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 229940039856 aricept Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000000133 brain stem Anatomy 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 235000021256 carbohydrate metabolism Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000006243 carbonyl protecting group Chemical group 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 230000004640 cellular pathway Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 231100000868 delusion Toxicity 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000009266 disease activity Effects 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 229960003530 donepezil Drugs 0.000 description 1
- 229940120402 donepezil and memantine Drugs 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000005183 environmental health Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 229940108366 exelon Drugs 0.000 description 1
- 208000019995 familial amyotrophic lateral sclerosis Diseases 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 229960003980 galantamine Drugs 0.000 description 1
- ASUTZQLVASHGKV-UHFFFAOYSA-N galanthamine hydrochloride Natural products O1C(=C23)C(OC)=CC=C2CN(C)CCC23C1CC(O)C=C2 ASUTZQLVASHGKV-UHFFFAOYSA-N 0.000 description 1
- 208000029364 generalized anxiety disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 208000035474 group of disease Diseases 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 208000011819 intense anxiety Diseases 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 208000028756 lack of coordination Diseases 0.000 description 1
- 229960004502 levodopa Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000008897 memory decline Effects 0.000 description 1
- 230000003923 mental ability Effects 0.000 description 1
- 230000003924 mental process Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000002025 microglial effect Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000004065 mitochondrial dysfunction Effects 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000008450 motivation Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 229940033872 namenda Drugs 0.000 description 1
- 229940053128 nerve growth factor Drugs 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000002241 neurite Anatomy 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 108060005714 orexin Proteins 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 208000019906 panic disease Diseases 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000008447 perception Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- HAGVCKULCLQGRF-UHFFFAOYSA-N pifithrin Chemical compound [Br-].C1=CC(C)=CC=C1C(=O)CN1[C+](N)SC2=C1CCCC2 HAGVCKULCLQGRF-UHFFFAOYSA-N 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 208000037821 progressive disease Diseases 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 229940051845 razadyne Drugs 0.000 description 1
- 239000012048 reactive intermediate Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000029964 regulation of glucose metabolic process Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 229960004136 rivastigmine Drugs 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 208000025874 separation anxiety disease Diseases 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 208000019116 sleep disease Diseases 0.000 description 1
- 208000022925 sleep disturbance Diseases 0.000 description 1
- 230000008454 sleep-wake cycle Effects 0.000 description 1
- 230000003997 social interaction Effects 0.000 description 1
- 230000008566 social perception Effects 0.000 description 1
- 101150062190 sod1 gene Proteins 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 201000001716 specific phobia Diseases 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 201000009032 substance abuse Diseases 0.000 description 1
- 231100000736 substance abuse Toxicity 0.000 description 1
- 208000011117 substance-related disease Diseases 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- JYTNQNCOQXFQPK-MRXNPFEDSA-N suvorexant Chemical compound C([C@H]1C)CN(C=2OC3=CC=C(Cl)C=C3N=2)CCN1C(=O)C1=CC(C)=CC=C1N1N=CC=N1 JYTNQNCOQXFQPK-MRXNPFEDSA-N 0.000 description 1
- 229960001198 suvorexant Drugs 0.000 description 1
- 230000009747 swallowing Effects 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 230000003976 synaptic dysfunction Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 230000001755 vocal effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 230000003936 working memory Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Biomedical Technology (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Hospice & Palliative Care (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Psychiatry (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present disclosure provides methods, compositions and kits for use in preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith as well as for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport.
Description
POLYPEPTIDES FOR USE IN THE TREATMENT AND PREVENTION OF
NEURODEGENERATIVE DISEASES
TECHNOLOGICAL FIELD
The present disclosure generally relates to polypeptides useful in the treatment, prevention and diagnosis of neurodegenerative diseases and conditions.
BACKGROUND ART
References considered to be relevant as background to the presently disclosed subject matter are listed below:
[1] WO 2006/046239.
NEURODEGENERATIVE DISEASES
TECHNOLOGICAL FIELD
The present disclosure generally relates to polypeptides useful in the treatment, prevention and diagnosis of neurodegenerative diseases and conditions.
BACKGROUND ART
References considered to be relevant as background to the presently disclosed subject matter are listed below:
[1] WO 2006/046239.
[2] WO 20 07/122622.
[3] WO 2007/091240.
[4] WO 2008/075349.
[5] Sandler, U. et al., 2010, Recent advances in clinical medicine, ISSN:
1790-5125.
1790-5125.
[6] Sandler, U. et al., 2010, J Experimental Therapeutics and Oncology 8:327-339.
[7] WO 2015/083167.
[8] WO 2017/134668.
Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
BACKGROUND OF THE INVENTION
Neurodegenerative diseases affect millions of people worldwide. Alzheimer's disease and Parkinson's disease are the most common neurodegenerative diseases. According to the National Institute of Environmental Health Sciences (NIH), in 2016, an estimated 5.4 million Americans were living with Alzheimer's disease.
Neurodegenerative diseases occur when nerve cells in the brain or peripheral nervous system lose function over time and ultimately die. While treatments may relieve some of the physical or mental symptoms associated with neurodegenerative diseases, there is currently no way to slow down disease progression and no known cures It is widely acceptable that the development of neurodegenerative diseases commences 10-20 years prior to their clinical manifestation. Although molecular mechanisms behind various neurodegenerative diseases are different, many processes, for example neurite retraction, dysfunction and destruction of synapses and ultimately neuronal death, are characteristic of neurodegeneration in general.
Furthermore, therapy in late stages of neurodegenerative diseases appears to be ineffective, most likely due to massive neuronal death, thus highlighting the importance of early detection and preventive measures.
A peptide termed "T101" was previously identified, as reported in the publication WO
2006/046239 (1). This peptide and derivatives thereof were suggested, inter cilia, for treatment of cancer. Treatment of cancer by using T101 was also suggested in the publication WO 2007/122622 (2), which demonstrates, among others, the effect of T101 on the development of various types of tumors. The peptide T101 was also described in the publication WO 2007/091240 (3) which relates, among others, to treatment of immunological diseases and in the publication WO
2008/075349 (4) as well as in the publications by Sandler et al. (5 and 6), relating to treating or preventing a disease involving a cell having T1/ST2 receptor.
In addition, an additional peptide derivative of T101, in which all of the amino acid residues are in their D configuration, has been previously reported to decrease the secretion of proteins that are known to be associated with cancer metastasis and to directly inhibit migration of cancer cells in vitro (7) This peptide derivative was further suggested for use in combination with additional anti-cancer agents, for the treatment of cancer (8).
SUMMARY OF THE INVENTION
In a first of its aspects, the present disclosure provides a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
In a second of its aspects, the present disclosure further provides a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
In a third of its aspects, the present disclosure provides a combination therapy comprising an isolated peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or a functional derivative thereof or a pharmaceutically acceptable salt of said isolated peptide and at least one additional therapeutic agent for use in a method of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
In a fourth of its aspects, the present disclosure provides a combination therapy comprising an isolated peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or a functional derivative thereof or a pharmaceutically acceptable salt of said isolated peptide and at least one additional therapeutic agent for use in improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
In yet a fifth of its aspects, the present disclosure provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated polypeptide or of a salt thereof.
By another sixth of its aspects, the present disclosure further provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated peptide or a salt thereof for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated peptide or of a salt thereof.
Still further another in a seventh of its aspects, the present disclosure provides a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent, for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
By a further eighth of its aspects, the present disclosure provides a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent, for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
In the ninth of its aspects, the present disclosure further provides a kit comprising:
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof, and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for at least one of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
Still further by a tenth of its aspects, the present disclosure provides a kit comprising:
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereoff, and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
Fig. 1A ¨ Fig. 1B are bar graphs showing in Fig. IA the level of the polypeptide dTCApFs (also termed herein "Nerofe", which is an all D amino acid polypeptide) in a brain faction of mice injected i.p. with dTCApFs (right bar) as compared to the level of the polypeptide in the brain of mice injected with 5% mannitol solution (left bar) and in Fig. 1B the level of a polypeptide having the sequence of dTCApFs though in all L amino acid residue configuration in a brain faction of mice injected i.p. with dTCApFs in all L amino acid residue configuration (right bar) as compared to the level of the polypeptide in the brain of mice injected with 5% mannitol solution (left bar).
Figure 2 is a graph showing the percentage of viable cells upon H202 insult at a concentration of 0.1 p,M (lower graph) and at a concentration of 0.01 1t1M-(higher graph) in cells incubated in the presence of dTCApFs at the indicated concentrations (iig/m1) Figure 3 is a graph showing expression of various genes in SH-SY5Y cells incubated in the presence of the indicated concentrations of dTCApFs (m/m1).
Figure 4 is a representative micrograph of a Western blot assay showing the expression level of Glut3 and Glutl in SHSY5Y cells under the indicated conditions (dTCApFs concentration, lig/m1).
Fig. 5A ¨ Fig. 5C show the effect of dTCApFs (indicated "D-NV) on the expression levels of the proteins XBP1 (Fig. 5A), p53 (Fig. 5B) and GLUT1 (Fig. 5C) in human neuroblastoma cells cells (SHSY5Y cells).
Fig. 6A ¨ Fig. 5C show the effect of a polypeptide having the amino acid sequence of dTCApFs though in all L amino acid configuration (indicated "NV) on the expression levels of the proteins XBP1 (Fig. 6A), p53 (Fig. 6B) and GLUT1 (Fig. 6C) in human neuroblastoma cells cells (SHSY5Y cells).
Figure 7 is a graph showing the percentage of viable cells upon H202 insult at a concentration of 0.1 mM in cells incubated in the presence of dTCApFs (upper graph) or L-amino aCTApFs (lower graph) at the indicated concentrations ( g/m1).
DETAILED DESCRIPTION OF EMBODIMENTS
The present disclosure is based on the surprising neuroprotective and therapeutic effects shown for the polypeptide dTCApFs, also termed herein "Nerofe", which has the amino acid sequence WWTFFLPSTLWERK in an all-D configuration (as denoted by SEQ ID NO:
1).
As shown in the Examples below, a good penetration ability of the BBB in mice was demonstrated for the polypeptide dTCApFs.
In addition, SH-SY5Y cells, which are often used as a model of neuronal function and differentiation (for example as means or studying Parkinson's disease) were used as a cellular model to study the effect of dTCApFs In this model, dTCApFs was shown to have a protective effect from hydrogen peroxide insults, in a dose dependent manner, as detailed in the appended Examples. It is of note that the protective effect on oxidative stress was better for the polypeptide dTCApFs in which all amino acid residues are all in the D configuration than for the corresponding polypeptide in which all the amino acid residues are in the L configuration.
Furthermore, when the effect of dTCApFs on gene expression in SH-SY5Y cells was examined, up regulation of the Superoxide dismutase 1 (SOD1) gene expression was observed.
Since the gene product of SOD1 assists in cell protection against oxidative stress, which is one of the manifestations of the development of neurodegenerative diseases and disorders, the above result provides further evidence to a protective effect to the cells by dTCApFs.
The effect of dTCApFs on expression of glucose transporters in human neurons was also examined by incubating SH-SY5Y cells in the presence of increasing doses of dTCApFs. As detailed below, the levels of Glucose Transporter 1 (GLUT1) and Glucose Transporter 3 (GLUT3) proteins were increased with the increase in dTCApFs doses, suggesting a further beneficial protective effect of the polypeptide on brain glucose metabolism and transportation, which are dramatically decreased in neurological disorders.
The effect of dTCApFs on expression of the )(BPI gene was further tested in SH-cells. XBP1 is an important transcription factor in human brain to which a beneficial effect on memory and cognitive capabilities is attributed. Surprisingly, expression of the XBP 1 protein was highly induced in the presence of the dTCApFs polypeptide.
Furthermore, incubation of the above cells in the presence of the dTCApFs polypeptide completely downregulated p53 expression in these cells. Lowering the expression level of p53 is considered to be a beneficial effect, since it leads to an increase in glucose metabolism.
Therefore, by a first aspect thereof, the present disclosure provides a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
In some embodiments, the method according to the present disclosure results in amelioration or reduction of at least one condition or symptom associated with at least one of a neurodegenerative disease and cognitive decline in a subject in need thereof In various embodiments the condition or symptom according to the present disclosure is at least one of anxiety, short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, impaired glucose metabolism, oxidative stress or any combination thereof.
In some embodiments, the method according to the present disclosure results in improvement of at least one of cognitive function, short term memory, long term memory, learning function and glucose metabolism and glucose transport in a subject in need thereof.
In some specific embodiments, the present disclosure provides a method for preventing or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof As it can be appreciated, the term "neurodegenerative disease" as used herein refers to a heterogeneous group of disorders that are characterized by the progressive degeneration of the structure and function of the central nervous system or the peripheral nervous system.
Any known neurodegenerative disease is encompassed by the present disclosure.
For example, but not limited to, Alzheimer's disease, Parkinson's disease, dementia (also known as "major neurocognitive disorder" and "mild neurocognitive disorder"), Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia or a disorder associated with protein misfolding, memory decline and anxiety, to name but few.
Mental disorders with neurodegenerative basis are also encompassed under the term neurodegenerative disease as herein defined, for example but not limited to, bipolar disorder.
Diagnosis of a neurodegenerative disease may be performed by a skilled physician according to methods well known in the art.
In the above and other embodiments the neurodegenerative disease according to the present disclosure is Alzheimer's disease, Parkinson's disease, Dementia, a disorder associated with protein misfolding, cognitive decline, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia, multiple sclerosis, Idiopathic Intracranial Hypertension, Cranial neuropathies, Trigeminal neuralgia, frontotemporal dementias (FTD), Senile Dementia (Dementia NOS), Motor Neuron Disease, or bipolar disorder.
In particular embodiments, the neurodegenerative disease according to the present disclosure is Al zheimer's disease.
Alzheimer's disease (AD) as known in the art relates to a progressive neurodegenerative disease that is manifested, among others, in damage and eventually destruction of brain cells, leading to memory loss and changes in cognitive and other brain functions.
More specifically, AD, refers to a disorder that involves deterioration of memory and other cognitive domains that leads to death within 3 to 9 years after diagnosis. The principal risk factor for Alzheimer's disease is
Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
BACKGROUND OF THE INVENTION
Neurodegenerative diseases affect millions of people worldwide. Alzheimer's disease and Parkinson's disease are the most common neurodegenerative diseases. According to the National Institute of Environmental Health Sciences (NIH), in 2016, an estimated 5.4 million Americans were living with Alzheimer's disease.
Neurodegenerative diseases occur when nerve cells in the brain or peripheral nervous system lose function over time and ultimately die. While treatments may relieve some of the physical or mental symptoms associated with neurodegenerative diseases, there is currently no way to slow down disease progression and no known cures It is widely acceptable that the development of neurodegenerative diseases commences 10-20 years prior to their clinical manifestation. Although molecular mechanisms behind various neurodegenerative diseases are different, many processes, for example neurite retraction, dysfunction and destruction of synapses and ultimately neuronal death, are characteristic of neurodegeneration in general.
Furthermore, therapy in late stages of neurodegenerative diseases appears to be ineffective, most likely due to massive neuronal death, thus highlighting the importance of early detection and preventive measures.
A peptide termed "T101" was previously identified, as reported in the publication WO
2006/046239 (1). This peptide and derivatives thereof were suggested, inter cilia, for treatment of cancer. Treatment of cancer by using T101 was also suggested in the publication WO 2007/122622 (2), which demonstrates, among others, the effect of T101 on the development of various types of tumors. The peptide T101 was also described in the publication WO 2007/091240 (3) which relates, among others, to treatment of immunological diseases and in the publication WO
2008/075349 (4) as well as in the publications by Sandler et al. (5 and 6), relating to treating or preventing a disease involving a cell having T1/ST2 receptor.
In addition, an additional peptide derivative of T101, in which all of the amino acid residues are in their D configuration, has been previously reported to decrease the secretion of proteins that are known to be associated with cancer metastasis and to directly inhibit migration of cancer cells in vitro (7) This peptide derivative was further suggested for use in combination with additional anti-cancer agents, for the treatment of cancer (8).
SUMMARY OF THE INVENTION
In a first of its aspects, the present disclosure provides a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
In a second of its aspects, the present disclosure further provides a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
In a third of its aspects, the present disclosure provides a combination therapy comprising an isolated peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or a functional derivative thereof or a pharmaceutically acceptable salt of said isolated peptide and at least one additional therapeutic agent for use in a method of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
In a fourth of its aspects, the present disclosure provides a combination therapy comprising an isolated peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or a functional derivative thereof or a pharmaceutically acceptable salt of said isolated peptide and at least one additional therapeutic agent for use in improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
In yet a fifth of its aspects, the present disclosure provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated polypeptide or of a salt thereof.
By another sixth of its aspects, the present disclosure further provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated peptide or a salt thereof for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated peptide or of a salt thereof.
Still further another in a seventh of its aspects, the present disclosure provides a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent, for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
By a further eighth of its aspects, the present disclosure provides a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent, for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
In the ninth of its aspects, the present disclosure further provides a kit comprising:
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof, and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for at least one of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
Still further by a tenth of its aspects, the present disclosure provides a kit comprising:
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereoff, and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
Fig. 1A ¨ Fig. 1B are bar graphs showing in Fig. IA the level of the polypeptide dTCApFs (also termed herein "Nerofe", which is an all D amino acid polypeptide) in a brain faction of mice injected i.p. with dTCApFs (right bar) as compared to the level of the polypeptide in the brain of mice injected with 5% mannitol solution (left bar) and in Fig. 1B the level of a polypeptide having the sequence of dTCApFs though in all L amino acid residue configuration in a brain faction of mice injected i.p. with dTCApFs in all L amino acid residue configuration (right bar) as compared to the level of the polypeptide in the brain of mice injected with 5% mannitol solution (left bar).
Figure 2 is a graph showing the percentage of viable cells upon H202 insult at a concentration of 0.1 p,M (lower graph) and at a concentration of 0.01 1t1M-(higher graph) in cells incubated in the presence of dTCApFs at the indicated concentrations (iig/m1) Figure 3 is a graph showing expression of various genes in SH-SY5Y cells incubated in the presence of the indicated concentrations of dTCApFs (m/m1).
Figure 4 is a representative micrograph of a Western blot assay showing the expression level of Glut3 and Glutl in SHSY5Y cells under the indicated conditions (dTCApFs concentration, lig/m1).
Fig. 5A ¨ Fig. 5C show the effect of dTCApFs (indicated "D-NV) on the expression levels of the proteins XBP1 (Fig. 5A), p53 (Fig. 5B) and GLUT1 (Fig. 5C) in human neuroblastoma cells cells (SHSY5Y cells).
Fig. 6A ¨ Fig. 5C show the effect of a polypeptide having the amino acid sequence of dTCApFs though in all L amino acid configuration (indicated "NV) on the expression levels of the proteins XBP1 (Fig. 6A), p53 (Fig. 6B) and GLUT1 (Fig. 6C) in human neuroblastoma cells cells (SHSY5Y cells).
Figure 7 is a graph showing the percentage of viable cells upon H202 insult at a concentration of 0.1 mM in cells incubated in the presence of dTCApFs (upper graph) or L-amino aCTApFs (lower graph) at the indicated concentrations ( g/m1).
DETAILED DESCRIPTION OF EMBODIMENTS
The present disclosure is based on the surprising neuroprotective and therapeutic effects shown for the polypeptide dTCApFs, also termed herein "Nerofe", which has the amino acid sequence WWTFFLPSTLWERK in an all-D configuration (as denoted by SEQ ID NO:
1).
As shown in the Examples below, a good penetration ability of the BBB in mice was demonstrated for the polypeptide dTCApFs.
In addition, SH-SY5Y cells, which are often used as a model of neuronal function and differentiation (for example as means or studying Parkinson's disease) were used as a cellular model to study the effect of dTCApFs In this model, dTCApFs was shown to have a protective effect from hydrogen peroxide insults, in a dose dependent manner, as detailed in the appended Examples. It is of note that the protective effect on oxidative stress was better for the polypeptide dTCApFs in which all amino acid residues are all in the D configuration than for the corresponding polypeptide in which all the amino acid residues are in the L configuration.
Furthermore, when the effect of dTCApFs on gene expression in SH-SY5Y cells was examined, up regulation of the Superoxide dismutase 1 (SOD1) gene expression was observed.
Since the gene product of SOD1 assists in cell protection against oxidative stress, which is one of the manifestations of the development of neurodegenerative diseases and disorders, the above result provides further evidence to a protective effect to the cells by dTCApFs.
The effect of dTCApFs on expression of glucose transporters in human neurons was also examined by incubating SH-SY5Y cells in the presence of increasing doses of dTCApFs. As detailed below, the levels of Glucose Transporter 1 (GLUT1) and Glucose Transporter 3 (GLUT3) proteins were increased with the increase in dTCApFs doses, suggesting a further beneficial protective effect of the polypeptide on brain glucose metabolism and transportation, which are dramatically decreased in neurological disorders.
The effect of dTCApFs on expression of the )(BPI gene was further tested in SH-cells. XBP1 is an important transcription factor in human brain to which a beneficial effect on memory and cognitive capabilities is attributed. Surprisingly, expression of the XBP 1 protein was highly induced in the presence of the dTCApFs polypeptide.
Furthermore, incubation of the above cells in the presence of the dTCApFs polypeptide completely downregulated p53 expression in these cells. Lowering the expression level of p53 is considered to be a beneficial effect, since it leads to an increase in glucose metabolism.
Therefore, by a first aspect thereof, the present disclosure provides a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
In some embodiments, the method according to the present disclosure results in amelioration or reduction of at least one condition or symptom associated with at least one of a neurodegenerative disease and cognitive decline in a subject in need thereof In various embodiments the condition or symptom according to the present disclosure is at least one of anxiety, short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, impaired glucose metabolism, oxidative stress or any combination thereof.
In some embodiments, the method according to the present disclosure results in improvement of at least one of cognitive function, short term memory, long term memory, learning function and glucose metabolism and glucose transport in a subject in need thereof.
In some specific embodiments, the present disclosure provides a method for preventing or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof As it can be appreciated, the term "neurodegenerative disease" as used herein refers to a heterogeneous group of disorders that are characterized by the progressive degeneration of the structure and function of the central nervous system or the peripheral nervous system.
Any known neurodegenerative disease is encompassed by the present disclosure.
For example, but not limited to, Alzheimer's disease, Parkinson's disease, dementia (also known as "major neurocognitive disorder" and "mild neurocognitive disorder"), Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia or a disorder associated with protein misfolding, memory decline and anxiety, to name but few.
Mental disorders with neurodegenerative basis are also encompassed under the term neurodegenerative disease as herein defined, for example but not limited to, bipolar disorder.
Diagnosis of a neurodegenerative disease may be performed by a skilled physician according to methods well known in the art.
In the above and other embodiments the neurodegenerative disease according to the present disclosure is Alzheimer's disease, Parkinson's disease, Dementia, a disorder associated with protein misfolding, cognitive decline, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia, multiple sclerosis, Idiopathic Intracranial Hypertension, Cranial neuropathies, Trigeminal neuralgia, frontotemporal dementias (FTD), Senile Dementia (Dementia NOS), Motor Neuron Disease, or bipolar disorder.
In particular embodiments, the neurodegenerative disease according to the present disclosure is Al zheimer's disease.
Alzheimer's disease (AD) as known in the art relates to a progressive neurodegenerative disease that is manifested, among others, in damage and eventually destruction of brain cells, leading to memory loss and changes in cognitive and other brain functions.
More specifically, AD, refers to a disorder that involves deterioration of memory and other cognitive domains that leads to death within 3 to 9 years after diagnosis. The principal risk factor for Alzheimer's disease is
9 age. The incidence of the disease doubles every 5 years after 65 years of age, however, up to 5%
of people with the disease have early onset AD (also known as younger-onset) that may appear at 40 or 50 years of age.
AD is a progressive disease, where dementia symptoms gradually worsen over several years. In its early stages, memory loss is mild, but with late-stage AD, individuals lose the ability to carry on a conversation and respond to their environment. Those with AD
live an average of eight years after their symptoms become noticeable to others, but survival can range from four to 20 years, depending on age and other health conditions.
The most common early symptom of AD is difficulty remembering newly learned information because AD changes typically begin in the part of the brain that affects learning. As AD advances through the brain it leads to increasingly severe symptoms, including disorientation, mood and behavior changes, deepening confusion about events, time and place, unfounded suspicions about family, friends and professional caregivers, more serious memory loss and behavior changes and difficulty speaking, swallowing and walking.
Beside symptomatic treatments to temporarily slow the worsening of dementia symptoms, AD has no current cure, and the current treatments cannot stop AD from progressing.
Diagnosis of AD may be performed by a skilled physician and include physical examinations and diagnostic tests (for example a Mini-Mental State Exam (MMSE)). Early signs and symptoms of Alzheimer's include among others memory impairment, difficulty concentrating, planning or problem-solving, problems with finishing daily tasks, confusion, language problems such as word-finding problems or reduced vocabulary in speech or writing, changes in mood, such as depression or other behavior and personality changes It should be appreciated that the polypeptide or a functional fragment or derivative thereof, salt thereof or any pharmaceutical composition comprising the same of the present disclosure are suitable for treating any stage of AD, at any age and for any conditions and symptoms associated therewith.
In other specific embodiments, as detailed above, the methods of the present disclosure are applicable, inter al/a, to Parkinson's disease or Dementia.
In particular embodiments, the neurodegenerative disease according to the present disclosure is Parkinson's disease.
As known in the art, Parkinson's disease is a progressive nervous system disorder that affects movement. Symptoms start gradually, sometimes starting with a barely noticeable tremor in just one hand. Tremors are common, but the disorder also commonly causes stiffness or slowing of movement. Parkinson's disease symptoms worsen as the condition progresses over time.
Currently, no cure is available for Parkinson's disease. However, medications might significantly improve the symptoms Parkinson's signs and symptoms may include tremor (or shaking, usually begins in a limb), slowed movement (bradykinesia), rigid muscles, impaired posture and balance, loss of automatic movements, speech changes, writing changes.
In further particular embodiments, the neurodegenerative disease according to the present disclosure is Dementia.
It should be appreciated that the term "Dementia" is used herein in a broad sense and refers to a broad category of brain diseases that cause a long-term and often gradual decrease in the ability to think and remember, severe to the extent of affecting daily functioning. The term "dementia" also encompasses major neurocognitive disorder and mild neurocognitive disorder.
Symptoms include emotional problems, difficulties with language and a decrease in motivation A diagnosis of dementia requires a change from a person's usual mental functioning and a greater decline than one would expect due to aging and is performed by a skilled physician.
In some embodiments, the method as herein defined is for preventing, treating, ameliorating, or delaying the onset of cognitive decline.
As known in the art, by the term "cognitive decline" as used herein it is referred to a gradual deterioration of mental faculties due to a neurological and/or psychological disturbance such as Alzheimer's disease, dementia, depression, or substance abuse.
In other words, the present disclosure provides a method for preventing, treating, ameliorating or delaying the onset of cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
In specific embodiment the present disclosure pertains to cognitive decline which is due to Alzheimer's disease.
As detailed above, the present disclosure provides a method for preventing, treating, ameliorating, or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
The terms "treat","treating","treatment" as used herein mean improving or reducing one or more clinical indicia of disease activity in a subject having a disease or disorder as herein defined. Improvement or reduction in the clinical indicia of disease may be subtle or significant.
The terms "disease" ,"disorder" or "condition" refer to a state in which there is a disturbance of normal functioning.
By the term "at least one" in the context of the "disease", "disorder" or "condition" as herein defined it is meant that a beneficial (therapeutic or prophylactic) effect may be achieved in at least one, for example 2, 3, 4, 5 or more diseases, disorders or conditions as herein defined, by administering the polypeptide, derivative or fragment thereof, salt thereof of the pharmaceutical composition comprising the same of the present disclosure.
Any condition or symptom associated with at least one of a neurodegenerative disease and cognitive decline in a subject in need thereof is encompassed by the present disclosure. Such symptoms (or conditions) generally include, but are not limited to, decline in mental abilities (for example short-term memory and long-term memory decline, impaired learning function), behavioral and psychiatric problems, lack of coordination, unsteady gait, speech changes, tremor, confusion with time or place, decreased or poor judgment, changes in mood and personality.
In some embodiments the method according to the present disclosure results in amelioration or reduction of at least one condition or symptom associated with at least one of a neurodegenerative disease and cognitive decline in a subject in need thereof.
By the term "amelioration" as used herein it is meant to reduce, alleviate, lighten, improve or relieve by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or about 100% at least one of the symptoms associated with at least one neurodegenerative disease and cognitive decline in a subject in need thereof.
The terms "ameliorating" and "reducing" are used herein interchangeably.
In specific embodiments the condition or symptom according to the present disclosure is at least one of anxiety, short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, impaired glucose metabolism, oxidative stress or any combination thereof.
As known in the art, experiencing occasional anxiety is a normal part of life.
However, people with anxiety (also referred to as "anxiety disorders") frequently have intense, excessive, and persistent worry and fear about everyday situations. Often, anxiety disorders involve repeated episodes of sudden feelings of intense anxiety and fear or terror that reach a peak within minutes (panic attacks). Examples of anxiety disorders include generalized anxiety disorder, social anxiety disorder (social phobia), specific phobias and separation anxiety disorder.
As known in the art, the term "short term memory" (also referred to as "primary" or "active memory") as used herein refers to the capacity for holding, but not manipulating, a small amount of information in mind in an active, readily available state for a short period of time. For example, short-term memory can be used to remember a phone number that has just been recited.
"Short-term memory impairment" as known in the art and as used herein refers to a situation when a person can remember incidents from 20 years ago but is fuzzy on the details of things that happened 20 minutes prior. It can cause people to forget the question they just asked or where they set their glasses down. Repetition of questions and behaviors is often a result of short-term memory impairment in dementia. There are a number of causes of short-term memory loss or impairment. some which are a result of medical conditions and others that are related to injuries or other outside influences. Short-term memory impairment is one of the earlier symptoms of for example Alzheimer's disease.
Long-term memory (LTM) is the stage where informative knowledge is held indefinitely.
It is defined in contrast to short-term and working memory, which persist for only about 18 to 30 seconds. Long-term memory loss or impairment as herein defined and as known in the art relates to a condition when a subject is experiencing trouble recalling information when he or she needs it. Long-term memory weakening is a normal part of aging.
By the term "cognitive function" as known in the art and as defined herein it is meant to relate to any mental process that involves symbolic operations, perception, memory, creation of imagery, and thinking and that encompasses awareness and capacity for judgment. "Impaired cognitive function" is when a person has trouble with cognitive function, for example as remembering, learning new things, concentrating, or making decisions that affect their everyday life. Cognitive impairment ranges from mild to severe. With mild impairment, people may begin to notice changes in cognitive functions, but still be able to do their everyday activities. Severe levels of impairment can lead to losing the ability to understand the meaning or importance of something and the ability to talk or write, resulting in the inability to live independently.
In some embodiments the impaired cognitive function includes impaired learning function.
The term "impaired learning function" as herein defined means a decrease or reduction in the learning function or learning ability, which may be defined for example as involving acquisition, organization, retention, understanding or use of verbal or nonverbal information. Impaired learning function results from impairments in one or more processes related to perceiving, thinking, remembering or learning, and may also involve difficulties with organizational skills, social perception, social interaction and perspective taking.
The present disclosure encompasses, among others, the neurodegenerative disease Mild cognitive impairment (MCI), which is the stage between the expected cognitive decline of normal aging and the more serious decline of dementia. MCI is characterized by problems with memory, language, thinking or judgment.
In various embodiments the method according to the present disclosure results in improvement of at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof Furthermore, the present disclosure provides a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:
1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
By the term "improvement" as used herein it is meant any recovery, advance or enhancement by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% of at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof By the term -preventing" it is meant to provide a "preventive treatment" or "prophylactic treatment", namely acting in a protective manner, defending against, or preventing something, especially a condition or disease as herein defined As known in the art, the disease, disorder, or condition as herein defined progress until they significantly impede the affected individual's quality of life By the term "delaying the onset" in the context of the disorder, disease or condition as defined herein, it is meant any postponement, suspension, impediment or retardation of the manifestation of the disease or symptoms associated therewith as herein defined.
In other embodiments, the functional fragment or derivative of the isolated polypeptide according to the present disclosure (namely the isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1) has at least 70% identity to the amino acid sequence denoted by SEQ
ID NO. 1.
In further embodiments, the isolated polypeptide according to the present disclosure consists of the amino acid sequence denoted by SEQ ID NO. 1.
In further embodiments, the isolated polypepti de according to the present disclosure is able to penetrate the Blood Brain Barrier.
In some embodiments, the method according to the present disclosure results in protection from oxidative stress in the neurons of said subject.
In some further embodiments, the peptide according to the present disclosure modulates the expression of at least one gene associated with protection from oxidative stress.
In some more specific embodiments, the at least one gene modulated by the peptide disclosed herein is any one of Superoxide dismutase 1 (SOD1), Brain-Derived Neurotrophic Factor (BDNF) and Tyrosine Hydroxylase (TH).
In some embodiments, the method according to the present disclosure method results in increasing glucose metabolism and/or transport in the neurons of said subject.
In some further embodiments, the peptide according to the present disclosure modulates the expression of at least one gene associated with glucose metabolism and/or transport.
In some more specific embodiments, the at least one gene modulated by the peptide disclosed herein is any one of GLUT1, GLUT3 and P53.
In some embodiments, the expression of the genes GLUT1 and GLUT3 is increased while the expression of the P53 gene is decreased by the peptide disclosed herein in the subject's neurons.
As detailed above the method according to the present disclosure results inter alia in improvement of glucose metabolism and glucose transport in a subject in need thereof.
By the term "glucose metabolism" (also referred to as "carbohydrate metabolism") as herein defined it is generally referred to oxidation, breakdown, and synthesis of carbohydrate in the living body's tissues. Glucose transporters are a wide group of membrane proteins that facilitate the transport of glucose across the plasma membrane, a process known inter alia as "facilitated diffusion". Since glucose is a vital source of energy for all life, these transporters are present in all phyla. Therefore, by the term "glucose transport- as herein defined it is referred to an entity associated with the transportation or transfer of carbohydrates in living organisms.
Glucose metabolism can be impaired by defects in insulin secretion from pancreatic beta cells or from defects in cellular sensitivity to insulin. It is well documented that impaired glucose metabolism or mitochondrial dysfunction is one of the major pathological changes observed in various neurodegenerative diseases including AD, Parkinson's disease (PD) or Huntington's disease (HD), thus suggesting that regulation of glucose metabolism and maintenance of mitochondrial homeostasis are critical for brain function.
By the term "oxidative stress" it is referred to an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA.
In humans, oxidative stress is thought to be involved in the development of various diseases, among which are attention deficit hyperactivity disorder (ADHD), cancer, Parkinson's disease, Lafora disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction and others.
In further embodiments, the method as herein defined comprises administering a pharmaceutical composition comprising said isolated polypeptide or a functional fragment or derivative of said isolated polypeptide or a salt thereof.
As detailed above, the present disclosure relates to methods of administering, uses of and pharmaceutical compositions comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof based on the surprising neuroprotective and therapeutic effects shown for the polypeptide dTCApFs (also termed herein "Nerofe").
The amino acid sequence of the polypeptide dTCApFs (Nerofe) is WWTFFLPSTLWERK
in a single-letter code and Trp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp Glu Arg Lys in three letter code, in which all of the amino acid residues are in their D
configuration. The amino acid sequence of the polypeptide dTCApFs is denoted herein by SEQ ID NO: 1. The polypeptide dTCApFs is the C-terminal 14 all D amino acid fragment of the "full T101 peptide" previously reported.
In other words, the present disclosure relates to use of an isolated polypeptide (interchangeably referred to herein as "peptide", "isolated peptide" or "isolated polypeptide") comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
As known in the art, a "polypeptide" refers to a molecular chain of amino acid residues, which can be optionally modified at one or more of its amino acid residues, for example by manosylation, glycosylation, amidation (for example C-terminal amides), carboxylation or phosphorylation. The polypeptide of the present disclosure may be obtained synthetically, through genetic engineering methods, expression in a host cell, or through any other suitable means as known in the art. Methods for producing peptides or polypeptides are well known in the art.
The terms "amino acid" or "amino acid residue" as used herein, refer to naturally occurring and synthetic amino acid residues, as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and 0-phosphoserine.
The term amino acid encompasses L-amino acids and D-amino acids, which are mirror images of L-amino acids, where the chirality at carbon alpha has been inverted. In particular, the polypeptide dTCApFs according to the present disclosure is an all D amino acid residue polypeptide, as detailed above.
The terms "amino acid sequence" or "polypeptide sequence" also relate to the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins The sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group.
The present disclosure further encompasses any pharmaceutically acceptable salt of said isolated polypeptide or peptide. The term "isolated" refers to molecules, such as the amino acid sequences described herein, peptides or polypeptides that are removed from their natural environment, isolated, or separated.
By the term "comprising" it is meant that the isolated polypeptide in accordance with the present disclosure includes the amino acid sequence denoted by SEQ ID NO: 1, as detailed above, but may also include additional amino acid residues at the N-terminus or at the C-terminus of the peptide or at both termini.
Furthermore in various embodiments the isolated polypeptide according to the present disclosure encompasses a polypeptide comprising the amino acid sequence denoted by SEQ ID
NO: 1, in which the N-terminus and/or the C-terminus of the peptide denoted by SEQ ID NO: 1 carries a protecting group. Protective (or protecting) groups are well known in the art and include inter cilia alcohol protecting groups, amine protecting groups, carbonyl protecting groups and others.
As indicated above, the present disclosure also encompasses a functional fragment or derivative of the isolated polypeptide as defined herein.
The term "fragment" as herein defined refers to any peptide or polypeptide which is at least one amino acid shorter than the isolated polypeptide in accordance with the present disclosure, obtained by deletion of at least one amino acid residue from the polypeptide in accordance with the present disclosure.
In some embodiments, a fragment of the isolated polypeptide in accordance with the present disclosure is a polypeptide that comprises a contiguous amino acid portion of SEQ ID NO:
1, that is 1, 2, 3, 4, 5 or more amino acid residues shorter than the sequence denoted by SEQ ID
NO: 1.
By the term "derivative" or "derivatives" it is meant to include polypeptides, which comprise the amino acid sequence denoted by SEQ 11) NO: 1, but differ in one or more amino acid residues in their overall sequence, namely, which have deletions, substitutions (e.g. replacement of at least one amino acid by another amino acid), inversions or additions within the overall sequence. This term also encompasses the replacement of at least one amino acid residue in the overall sequence by its respective L amino acid residue.
Derivatives also encompass amino acid sequence denoted by SEQ ID NO: 1, in which at least one amino acid residue is replaced by a synthetic amino acid residue, an amino acid analogue or an amino acid mimetic.
As known in the art, amino acid "substitutions" are the result of replacing one amino acid with another amino acid, for example with another amino acid that has similar structural and/or chemical properties (conservative amino acid replacements). Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, each of the following eight groups contains amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).
It is appreciated that the fragment or derivative of the isolated polypeptide as defined herein must not alter the biological activity of the original polypeptide. By the term "functional' it is meant to encompass any fragment or derivative of the amino acid sequence denoted by SEQ ID
NO: 1, which retains a biological activity qualitatively similar to that of the unmodified polypeptide (namely a polypeptide having the amino acid sequence denoted by SEQ ID NO: 1.
The biological activity of the fragment or derivative as herein defined may be determined by any method known in the art, for example by monitoring the effect of administering the polypeptide as herein defined to model cells or animals In particular embodiments, the present disclosure relates to a functional fragment or derivative of the isolated polypeptide comprising amino acid sequence denoted by SEQ ID NO. 1, wherein said functional fragment or derivative has an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, more preferably 95%, in particular 96%, 97%, 98% or 99% identical to the amino acid sequence of the unmodified isolated polypeptide of the present disclosure, namely to one of the amino acid sequences denoted by SEQ ID NO: 1.
In specific embodiments, the functional fragment or derivative of the polypeptide as herein defined has at least 70% identity to the amino acid sequence denoted by SEQ ID
NO. 1. In further specific embodiments, the polypeptide as herein defined consists of the amino acid sequence denoted by SEQ ID NO. 1.
It should be appreciated that the terms "identical", or percent "identity", in the context of two or more amino acids or nucleic acids sequences, refer to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70%, identity, 75% identity, preferably 80%, 85%, 90%, or higher identity) over a specified region, when compared and aligned for maximum correspondence over a comparison window.
The peptide of the present disclosure may be administered per se or in combination with another active agent.
Thus in various further embodiments, the method as herein defined further comprises administering at least one additional therapeutic agent to said subject.
By the term "additional therapeutic agent" in the context of the present disclosure it is referred to any therapeutic agent known to a skilled physician for the treatment or prevention of a neurodegenerative disease. For example, Aricept , Exelon , Razadyne , Memantine, Namenda or Levodopa, to name but few, may be used as additional therapeutic agents for the treatment of the respective neurodegenerative disease as further detailed below.
In some embodiments, the subj ect suitable for the methods of the present disclosure is not suffering from any type of cancer. In some specific embodiments, the subject is not suffering from brain cancer, e.g. neuroblastoma.
All the embodiments as described above and further below, concerning the uses, compositions and kits of the isolated peptide according to the present disclosure are relevant to all the different aspects disclosed herein.
In a second of its aspects, the present disclosure further provides a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypepti de or a salt thereof.
In some embodiments the peptide of the present disclosure may be administered in a pharmaceutical composition along with a pharmaceutically acceptable excipient, carrier, or diluent.
Administration of the isolated polypeptide or a functional fragment or derivative thereof or a pharmaceutical composition comprising the same according to the present disclosure may be performed by any one of the acceptable routes, for example but not limited to oral administration, intravenous, intramuscular, intraperitoneal, intrathecal or subcutaneous injection, intra-rectal admini strati on, intranas al administration, ocular administration or topical administration.
As mentioned above, the functional fragment or derivative according to the present disclosure has at least 70% identity to the amino acid sequence denoted by SEQ
ID NO. 1.
In some more specific embodiments, the polypeptide consists of the amino acid sequence denoted by SEQ ID NO. 1.
In a further aspect, the present disclosure provides a combination therapy comprising an isolated peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or a functional derivative thereof or a pharmaceutically acceptable salt of said isolated peptide and at least one additional therapeutic agent for use in a method of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
In another aspect, the present disclosure provides a combination therapy comprising an isolated peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or a functional derivative thereof or a pharmaceutically acceptable salt of said isolated peptide and at least one additional therapeutic agent for use in improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
In some embodiments, the at least one additional therapeutic agent may refer to any therapeutic agent known to a skilled physician for the treatment or prevention of Alzheimer's disease.
Although current medications cannot cure Alzheimer's, some treatments address the underlying biology. Other medications may help lessen symptoms, such as memory loss and confusion. To date, the U.S. Food and Drug Administration (FDA) has approved medications that fall into two categories: drugs that may change disease progression in people living with Alzheimer's, and drugs that may temporarily mitigate some symptoms of Alzheimer's disease.
The drugs that may change disease progression may provide benefits to both cognition and function in people living with Alzheimer's disease For example, Aducanumab (AduhelmTM) is an anti-amyloid antibody intravenous (IV) infusion therapy approved for Alzheimer's disease. In some embodiments, the at least one additional therapeutic agent may be Aducanumab (AduhelmTm).
Among the drugs that treat symptoms, there are some for cognitive symptoms and others for non-cognitive symptoms (memory and thinking).
While medications that treat cognitive symptoms do not stop the damage Alzheimer's causes to brain cells, they may help lessen or stabilize symptoms for a limited time by affecting certain chemicals between the brain's nerve cells.
The following medications are non-limiting examples of drugs prescribed to treat symptoms related to memory and thinking.
In some embodiments, the at least one additional therapeutic agent may be a cholinesterase inhibitor.
Cholinesterase inhibitors are prescribed to treat symptoms related to memory, thinking, language, judgment and other thought processes. These medications prevent the breakdown of acetylcholine, a chemical messenger important for memory and learning. These drugs support communication between nerve cells.
Non-limiting examples of cholinesterase inhibitors suitable according to the present disclosure are: Donepezil (Aricept0) approved to treat all stages of Alzheimer's disease, Rivastigmine (Exelon0) approved for mild-to-moderate Alzheimer's as well as mild-to-moderate dementia associated with Parkinson's disease and Galantamine (Razadyneg) approved for mild-to-moderate stages of Alzheimer's disease.
In some embodiments, the at least one additional therapeutic agent may be a glutamate regulator.
Glutamate regulators are prescribed to improve memory, attention, reason, language and the ability to perform simple tasks. This type of drug works by regulating the activity of glutamate, a different chemical messenger. For example, Memantine (Namenda0) was approved for moderate-to-severe Alzheimer' s disease.
Furthermore, an additional exemplary treatment may be a combination of a cholinesterase inhibitor and a glutamate regulator such as Donepezil and memantine (Namzaric0) approved for moderate-to-severe Alzheimer's disease.
An example of medications for treating non-cognitive symptoms of Alzheimer's disease i.e. behavioral and psychological symptoms such as sleep disturbances, agitation, hallucinations and delusions may be an Orexin receptor antagonist. Orexin receptor antagonist such as Suvorexant (Belsomra0) is prescribed to treat insomnia for individuals living with dementia, this drug is thought to inhibit the activity of orexin, a type of neurotransmitter involved in the sleep-wake cycle and is approved for mild-to-moderate Alzheimer's disease.
In some embodiments, the at least one additional therapeutic agent suitable for the combination therapy disclosed herein is any one of cholinesterase inhibitors, glutamate regulators, a combination of a cholinesterase inhibitor and a glutamate regulators, Orexin receptor antagonist or Aducanumab.
In certain embodiments, the isolated peptide and said additional therapeutic agent are administered concomitantly or consecutively.
Still further in an additional aspect, the present disclosure provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated polypeptide or of a salt thereof.
In some embodiments, the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is wherein the method results in amelioration or reduction of at least one condition or symptom associated with at least one of a neurodegenerative disease and cognitive decline in a subject in need thereof.
In other embodiments, the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is wherein the condition or symptom is at least one of anxiety, short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, impaired glucose metabolism, oxidative stress or any combination thereof.
In further embodiments the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is wherein the method results in improvement of at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof In some embodiments, the isolated polypeptide according to the present disclosure is for use in a method for preventing or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
In further embodiments, the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is for preventing, treating, ameliorating, or delaying the onset of cognitive decline.
As mentioned above, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Dementia, a disorder associated with protein misfolding, cognitive decline, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MIND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Fri edreich's Ataxia, multiple sclerosis, Idiopathic Intracranial Hypertension, Cranial neuropathies, Trigeminal neuralgia, frontotemporal dementias (FTD), Senile Dementia (Dementia NOS), Motor Neuron Disease, or bipolar disorder.
In various embodiments the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is applicable to Alzheimer's disease, Parkinson's disease, Dementia or cognitive decline.
In some embodiments, neurodegenerative disease is Alzheimer's disease.
In certain embodiments, the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is for preventing, treating, ameliorating, or delaying the onset of cognitive decline.
In some embodiments, the functional fragment or derivative of the isolated peptide as disclosed herein has at least 70% identity to the amino acid sequence denoted by SEQ lD NO. 1.
In certain embodiments, the polypeptide consists of the amino acid sequence denoted by SEQ ID NO. 1.
In some further embodiments, the polypeptide is able to penetrate the Blood Brain Barrier.
As mentioned above, the isolated peptide as disclosed herein is for use in a method that results in protection from oxidative stress in the neurons of said subject.
In some specific embodiments, the isolated peptide modulates the expression of at least one gene associated with protection from oxidative stress In certain embodiments, the at least one gene is any one of SOD1, BDNF and TH.
As mentioned above, the isolated peptide as disclosed herein is for use in a method that results in increasing glucose metabolism and/or transport in the neurons of said subject.
In some specific embodiments, the isolated peptide modulates the expression of at least one gene associated with glucose metabolism and/or transport.
In certain embodiments, the at least one gene is any one of GLUT1, GLUT3 and P53.
As mentioned above, the isolated peptide as disclosed herein is for use in a method comprising administering a pharmaceutical composition comprising said isolated polypeptide or a functional fragment or derivative of said isolated peptide or a salt thereof.
In some further embodiments, the isolated peptide as disclosed herein is for use in a method further comprising administering at least one additional therapeutic agent to said subject.
In a further aspect, the present disclosure provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated peptide or a salt thereof for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated peptide or of a salt thereof.
As mentioned above in some embodiments, the functional fragment or derivative of the isolated peptide as disclosed herein has at least 70% identity to the amino acid sequence denoted by SEQ ID NO. 1. In certain embodiments, the polypeptide consists of the amino acid sequence denoted by SEQ ID NO. 1.
Still further the present disclosure provides a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent, for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
In some embodiments, the pharmaceutical composition comprising the isolated peptide as disclosed herein is use in a method for preventing or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
By a further aspect thereof the present disclosure provides a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ
ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent, for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof The term "pharnuzceutical composition" as herein defined generally comprises as an active agent an isolated polypeptide according to the present disclosure (namely an isolated polypeptide comprising or consisting of the amino acid sequence denoted by SEQ
ID NO: 1) or a functional fragment or derivative of the isolated polypeptide or a salt thereof, and at least one of a buffering agent, an agent which adjusts the osmolarity thereof, and optionally, at least one pharmaceutically acceptable excipient, carrier, diluent and/or additive as known in the art.
As used herein the term "pharmaceutically acceptable excipient, carrier, diluent and/or additive" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like, as known in the art. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.
The pharmaceutical compositions according to the invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutically acceptable carrier(s), excipient(s) or additive(s).
For example, the term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents as well known in the art. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
The pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
Supplementary or additive active ingredients, for example additional anti-cancer agents, can also be incorporated into the pharmaceutical composition of the invention.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
By the term "effective amount" as used herein it is meant an amount necessary to achieve a desirable result. The effective amount is determined by the severity and type of the disease or condition in conjunction with the preventive or therapeutic objectives, the route of administration and the patient's general condition (age, sex, weight and other considerations known to the attending physician). The effective amount may be determined based on animal models and is determined by a skilled physician.
By the term "subject in need thereof' or "subject" as used herein it is meant to include warm-blooded animals (such as for example humans, rats, mice, dogs, cats, guinea pigs and primates). In particular embodiments, the subject is human.
In some embodiments the subject according to the present disclosure is diagnosed by a skilled physician as being at a risk of developing a neurodegenerative disease or cognitive decline.
In some other embodiments the subject is diagnosed as having the disease, disorder or condition as herein defined. Diagnosis of the disease, disorder or condition as herein defined may be performed by a skilled physician, by means and steps as known in the art.
In certain embodiments, the polypeptide of the pharmaceutical composition for use according to the present disclosure consists of the amino acid sequence denoted by SEQ ID NO.
1.
In some particular embodiments, the neurodegenerative disorder is Alzheimer's disease In another aspect, the present disclosure further provides a kit comprising:
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof; and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for at least one of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
A further aspect of the present disclosure provides a kit comprising:
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof; and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
The kit according to the present disclosure may further include instructions for use.
The isolated polypeptide according to the present disclosure or a functional fragment or derivative of the isolated polypeptide or a salt thereof and the at least one pharmaceutically acceptable excipient, carrier or diluent may be contained in the same container or in different ones.
Still further, the kit according to the present disclosure may include container means for containing separate kit components. In some embodiments the kit of the present disclosure further includes at least one solvent or buffer known in the art.
The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20%
higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. As used herein the term "about" refers to 10 %.
The terms "comprise", "comprises", "comprising","includes","including", "having" and their conjugates mean "including but not limited to". This term encompasses the term "consisting of". Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises"
and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. The following examples are representative of techniques employed by the Inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.
It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.
Experimental procedures Mice ICR female mice having initial body weight of 20-22 grams were used (Envigo).
Blood brain barrier (BBB) penetration assay A control group of 6 mice was injected intraperitoneally (I.P.) with 5%
mannitol solution and another group of 6 mice was injected I.P. with the polypeptide dTCApFs (also termed herein Nerofe, at 60 mg/kg) in a 5% mannitol solution. Spinal fluid was collected after the injection (1 hour) and subjected to ELISA analysis of dTCApFs (Nerofe), as detailed below.
ELISA analysis of niice cerebrospinal fluids Mice were injected with 60 mg/kg D-amino acid dTCApFs (Nerofe), and cerebrospinal fluid (about 10 pl) and serum were collected after 1 hr, and frozen. A
calibration curve was prepared by serial dilutions of the polypeptide in PBS (Biological industries, 02-023-5A) as noted in table 1 below.
Next, the cerebrospinal samples were thawed and kept on ice, and then diluted in 220 ill PBS. Sample loading was next performed by loading 100 ul duplicates of each sample and of the standard in a Maxisorp 96-wells plate (NUNC, F96 Maxisorp, 442404). The plate was sealed and incubated at 4 C overnight with gentle shaking. The plate was washed by removing the liquid and washing the plate four (4) times using a multi-pipette or an automated washer with 300 41 0.05%
TW-20 (Amresco, 0777-1L) in PBS. The samples were blocked by diluting 5% BSA
(MP
biomedicals, 160069) in PBS. Then, 300 ul of the blocking buffer were loaded in each well and the plate was incubated at room temperature (R.T.) for 1 hour with gentle shaking. The Plates were washed again, as described above.
Detection was performed by diluting the first antibody according to table 1 below in diluent (0.05% TW-20, 0.1% BSA in PBS). Then, 100 ul of detection antibody were loaded into each well and the plate was incubated at R.T. for 2:00 hours with gentle shaking. The plate was washed again as detailed above. Next, FIRP conjugate was prepared by diluting goat anti-rabbit HRP conjugate antibody (Cell signaling, 7074) according to table 1 in diluent. HRP conjugate (100 pi) was then loaded into each well and the plate was incubated for 1:00 hour at R.T. with gentle shaking. The plate was washed again, as detailed above.
Development was performed by adding 100 pl TMB (Milipore, ES001-500ML) to each well, and waiting for blue color development, then adding 100 ul 2N H2SO4 (Frutarom, 5552540).
Plate reading was performed in a microplate reader, by checking the wells absorbance at 450 nm.
Table 1 Calibration curve preparation Peptide Calibration curve 1st antibody dilution Secondary antibody range dilution dTCApFs 1000-15.6 pg/ml 1:250 1:300 Preparation of the dTCApFs peptide and compositions comprising thereof The polypeptide termed herein dTCApFs (or Nerofe, both terms are used herein interchangeably and refer to the same peptide as indicated above) is a 14 amino acid residues long peptide, in which all of the amino acid residues are at their D configuration, having the amino acid sequence of Trp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp Glu Arg Lys (or WWTFFLPSTLWERK
in a single letter code, as denoted by SEQ ID NO: 1). A peptide having an amino acid sequence of Trp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp Glu Arg Lys (or WWTFFLPSTLWERK in a single letter code, as denoted by SEQ ID NO: 2) in which all of the amino acid residues are at their L
configuration, was also used. The above D-amino acid peptide was GlVIP
manufactured in BCN
peptides at Barcelona. The above L-amino acid peptide was manufactured at Anaspec Inc., in the USA.
Cell viability assay (Resazurin) of cells treated with dTCApFs (Nerofe) Materials:
Clear bottom 96 well cell culture plates (Greiner 60-65590) Cell Culture Media:
of people with the disease have early onset AD (also known as younger-onset) that may appear at 40 or 50 years of age.
AD is a progressive disease, where dementia symptoms gradually worsen over several years. In its early stages, memory loss is mild, but with late-stage AD, individuals lose the ability to carry on a conversation and respond to their environment. Those with AD
live an average of eight years after their symptoms become noticeable to others, but survival can range from four to 20 years, depending on age and other health conditions.
The most common early symptom of AD is difficulty remembering newly learned information because AD changes typically begin in the part of the brain that affects learning. As AD advances through the brain it leads to increasingly severe symptoms, including disorientation, mood and behavior changes, deepening confusion about events, time and place, unfounded suspicions about family, friends and professional caregivers, more serious memory loss and behavior changes and difficulty speaking, swallowing and walking.
Beside symptomatic treatments to temporarily slow the worsening of dementia symptoms, AD has no current cure, and the current treatments cannot stop AD from progressing.
Diagnosis of AD may be performed by a skilled physician and include physical examinations and diagnostic tests (for example a Mini-Mental State Exam (MMSE)). Early signs and symptoms of Alzheimer's include among others memory impairment, difficulty concentrating, planning or problem-solving, problems with finishing daily tasks, confusion, language problems such as word-finding problems or reduced vocabulary in speech or writing, changes in mood, such as depression or other behavior and personality changes It should be appreciated that the polypeptide or a functional fragment or derivative thereof, salt thereof or any pharmaceutical composition comprising the same of the present disclosure are suitable for treating any stage of AD, at any age and for any conditions and symptoms associated therewith.
In other specific embodiments, as detailed above, the methods of the present disclosure are applicable, inter al/a, to Parkinson's disease or Dementia.
In particular embodiments, the neurodegenerative disease according to the present disclosure is Parkinson's disease.
As known in the art, Parkinson's disease is a progressive nervous system disorder that affects movement. Symptoms start gradually, sometimes starting with a barely noticeable tremor in just one hand. Tremors are common, but the disorder also commonly causes stiffness or slowing of movement. Parkinson's disease symptoms worsen as the condition progresses over time.
Currently, no cure is available for Parkinson's disease. However, medications might significantly improve the symptoms Parkinson's signs and symptoms may include tremor (or shaking, usually begins in a limb), slowed movement (bradykinesia), rigid muscles, impaired posture and balance, loss of automatic movements, speech changes, writing changes.
In further particular embodiments, the neurodegenerative disease according to the present disclosure is Dementia.
It should be appreciated that the term "Dementia" is used herein in a broad sense and refers to a broad category of brain diseases that cause a long-term and often gradual decrease in the ability to think and remember, severe to the extent of affecting daily functioning. The term "dementia" also encompasses major neurocognitive disorder and mild neurocognitive disorder.
Symptoms include emotional problems, difficulties with language and a decrease in motivation A diagnosis of dementia requires a change from a person's usual mental functioning and a greater decline than one would expect due to aging and is performed by a skilled physician.
In some embodiments, the method as herein defined is for preventing, treating, ameliorating, or delaying the onset of cognitive decline.
As known in the art, by the term "cognitive decline" as used herein it is referred to a gradual deterioration of mental faculties due to a neurological and/or psychological disturbance such as Alzheimer's disease, dementia, depression, or substance abuse.
In other words, the present disclosure provides a method for preventing, treating, ameliorating or delaying the onset of cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
In specific embodiment the present disclosure pertains to cognitive decline which is due to Alzheimer's disease.
As detailed above, the present disclosure provides a method for preventing, treating, ameliorating, or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
The terms "treat","treating","treatment" as used herein mean improving or reducing one or more clinical indicia of disease activity in a subject having a disease or disorder as herein defined. Improvement or reduction in the clinical indicia of disease may be subtle or significant.
The terms "disease" ,"disorder" or "condition" refer to a state in which there is a disturbance of normal functioning.
By the term "at least one" in the context of the "disease", "disorder" or "condition" as herein defined it is meant that a beneficial (therapeutic or prophylactic) effect may be achieved in at least one, for example 2, 3, 4, 5 or more diseases, disorders or conditions as herein defined, by administering the polypeptide, derivative or fragment thereof, salt thereof of the pharmaceutical composition comprising the same of the present disclosure.
Any condition or symptom associated with at least one of a neurodegenerative disease and cognitive decline in a subject in need thereof is encompassed by the present disclosure. Such symptoms (or conditions) generally include, but are not limited to, decline in mental abilities (for example short-term memory and long-term memory decline, impaired learning function), behavioral and psychiatric problems, lack of coordination, unsteady gait, speech changes, tremor, confusion with time or place, decreased or poor judgment, changes in mood and personality.
In some embodiments the method according to the present disclosure results in amelioration or reduction of at least one condition or symptom associated with at least one of a neurodegenerative disease and cognitive decline in a subject in need thereof.
By the term "amelioration" as used herein it is meant to reduce, alleviate, lighten, improve or relieve by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or about 100% at least one of the symptoms associated with at least one neurodegenerative disease and cognitive decline in a subject in need thereof.
The terms "ameliorating" and "reducing" are used herein interchangeably.
In specific embodiments the condition or symptom according to the present disclosure is at least one of anxiety, short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, impaired glucose metabolism, oxidative stress or any combination thereof.
As known in the art, experiencing occasional anxiety is a normal part of life.
However, people with anxiety (also referred to as "anxiety disorders") frequently have intense, excessive, and persistent worry and fear about everyday situations. Often, anxiety disorders involve repeated episodes of sudden feelings of intense anxiety and fear or terror that reach a peak within minutes (panic attacks). Examples of anxiety disorders include generalized anxiety disorder, social anxiety disorder (social phobia), specific phobias and separation anxiety disorder.
As known in the art, the term "short term memory" (also referred to as "primary" or "active memory") as used herein refers to the capacity for holding, but not manipulating, a small amount of information in mind in an active, readily available state for a short period of time. For example, short-term memory can be used to remember a phone number that has just been recited.
"Short-term memory impairment" as known in the art and as used herein refers to a situation when a person can remember incidents from 20 years ago but is fuzzy on the details of things that happened 20 minutes prior. It can cause people to forget the question they just asked or where they set their glasses down. Repetition of questions and behaviors is often a result of short-term memory impairment in dementia. There are a number of causes of short-term memory loss or impairment. some which are a result of medical conditions and others that are related to injuries or other outside influences. Short-term memory impairment is one of the earlier symptoms of for example Alzheimer's disease.
Long-term memory (LTM) is the stage where informative knowledge is held indefinitely.
It is defined in contrast to short-term and working memory, which persist for only about 18 to 30 seconds. Long-term memory loss or impairment as herein defined and as known in the art relates to a condition when a subject is experiencing trouble recalling information when he or she needs it. Long-term memory weakening is a normal part of aging.
By the term "cognitive function" as known in the art and as defined herein it is meant to relate to any mental process that involves symbolic operations, perception, memory, creation of imagery, and thinking and that encompasses awareness and capacity for judgment. "Impaired cognitive function" is when a person has trouble with cognitive function, for example as remembering, learning new things, concentrating, or making decisions that affect their everyday life. Cognitive impairment ranges from mild to severe. With mild impairment, people may begin to notice changes in cognitive functions, but still be able to do their everyday activities. Severe levels of impairment can lead to losing the ability to understand the meaning or importance of something and the ability to talk or write, resulting in the inability to live independently.
In some embodiments the impaired cognitive function includes impaired learning function.
The term "impaired learning function" as herein defined means a decrease or reduction in the learning function or learning ability, which may be defined for example as involving acquisition, organization, retention, understanding or use of verbal or nonverbal information. Impaired learning function results from impairments in one or more processes related to perceiving, thinking, remembering or learning, and may also involve difficulties with organizational skills, social perception, social interaction and perspective taking.
The present disclosure encompasses, among others, the neurodegenerative disease Mild cognitive impairment (MCI), which is the stage between the expected cognitive decline of normal aging and the more serious decline of dementia. MCI is characterized by problems with memory, language, thinking or judgment.
In various embodiments the method according to the present disclosure results in improvement of at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof Furthermore, the present disclosure provides a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:
1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
By the term "improvement" as used herein it is meant any recovery, advance or enhancement by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% of at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof By the term -preventing" it is meant to provide a "preventive treatment" or "prophylactic treatment", namely acting in a protective manner, defending against, or preventing something, especially a condition or disease as herein defined As known in the art, the disease, disorder, or condition as herein defined progress until they significantly impede the affected individual's quality of life By the term "delaying the onset" in the context of the disorder, disease or condition as defined herein, it is meant any postponement, suspension, impediment or retardation of the manifestation of the disease or symptoms associated therewith as herein defined.
In other embodiments, the functional fragment or derivative of the isolated polypeptide according to the present disclosure (namely the isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1) has at least 70% identity to the amino acid sequence denoted by SEQ
ID NO. 1.
In further embodiments, the isolated polypeptide according to the present disclosure consists of the amino acid sequence denoted by SEQ ID NO. 1.
In further embodiments, the isolated polypepti de according to the present disclosure is able to penetrate the Blood Brain Barrier.
In some embodiments, the method according to the present disclosure results in protection from oxidative stress in the neurons of said subject.
In some further embodiments, the peptide according to the present disclosure modulates the expression of at least one gene associated with protection from oxidative stress.
In some more specific embodiments, the at least one gene modulated by the peptide disclosed herein is any one of Superoxide dismutase 1 (SOD1), Brain-Derived Neurotrophic Factor (BDNF) and Tyrosine Hydroxylase (TH).
In some embodiments, the method according to the present disclosure method results in increasing glucose metabolism and/or transport in the neurons of said subject.
In some further embodiments, the peptide according to the present disclosure modulates the expression of at least one gene associated with glucose metabolism and/or transport.
In some more specific embodiments, the at least one gene modulated by the peptide disclosed herein is any one of GLUT1, GLUT3 and P53.
In some embodiments, the expression of the genes GLUT1 and GLUT3 is increased while the expression of the P53 gene is decreased by the peptide disclosed herein in the subject's neurons.
As detailed above the method according to the present disclosure results inter alia in improvement of glucose metabolism and glucose transport in a subject in need thereof.
By the term "glucose metabolism" (also referred to as "carbohydrate metabolism") as herein defined it is generally referred to oxidation, breakdown, and synthesis of carbohydrate in the living body's tissues. Glucose transporters are a wide group of membrane proteins that facilitate the transport of glucose across the plasma membrane, a process known inter alia as "facilitated diffusion". Since glucose is a vital source of energy for all life, these transporters are present in all phyla. Therefore, by the term "glucose transport- as herein defined it is referred to an entity associated with the transportation or transfer of carbohydrates in living organisms.
Glucose metabolism can be impaired by defects in insulin secretion from pancreatic beta cells or from defects in cellular sensitivity to insulin. It is well documented that impaired glucose metabolism or mitochondrial dysfunction is one of the major pathological changes observed in various neurodegenerative diseases including AD, Parkinson's disease (PD) or Huntington's disease (HD), thus suggesting that regulation of glucose metabolism and maintenance of mitochondrial homeostasis are critical for brain function.
By the term "oxidative stress" it is referred to an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA.
In humans, oxidative stress is thought to be involved in the development of various diseases, among which are attention deficit hyperactivity disorder (ADHD), cancer, Parkinson's disease, Lafora disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction and others.
In further embodiments, the method as herein defined comprises administering a pharmaceutical composition comprising said isolated polypeptide or a functional fragment or derivative of said isolated polypeptide or a salt thereof.
As detailed above, the present disclosure relates to methods of administering, uses of and pharmaceutical compositions comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof based on the surprising neuroprotective and therapeutic effects shown for the polypeptide dTCApFs (also termed herein "Nerofe").
The amino acid sequence of the polypeptide dTCApFs (Nerofe) is WWTFFLPSTLWERK
in a single-letter code and Trp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp Glu Arg Lys in three letter code, in which all of the amino acid residues are in their D
configuration. The amino acid sequence of the polypeptide dTCApFs is denoted herein by SEQ ID NO: 1. The polypeptide dTCApFs is the C-terminal 14 all D amino acid fragment of the "full T101 peptide" previously reported.
In other words, the present disclosure relates to use of an isolated polypeptide (interchangeably referred to herein as "peptide", "isolated peptide" or "isolated polypeptide") comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
As known in the art, a "polypeptide" refers to a molecular chain of amino acid residues, which can be optionally modified at one or more of its amino acid residues, for example by manosylation, glycosylation, amidation (for example C-terminal amides), carboxylation or phosphorylation. The polypeptide of the present disclosure may be obtained synthetically, through genetic engineering methods, expression in a host cell, or through any other suitable means as known in the art. Methods for producing peptides or polypeptides are well known in the art.
The terms "amino acid" or "amino acid residue" as used herein, refer to naturally occurring and synthetic amino acid residues, as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and 0-phosphoserine.
The term amino acid encompasses L-amino acids and D-amino acids, which are mirror images of L-amino acids, where the chirality at carbon alpha has been inverted. In particular, the polypeptide dTCApFs according to the present disclosure is an all D amino acid residue polypeptide, as detailed above.
The terms "amino acid sequence" or "polypeptide sequence" also relate to the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins The sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group.
The present disclosure further encompasses any pharmaceutically acceptable salt of said isolated polypeptide or peptide. The term "isolated" refers to molecules, such as the amino acid sequences described herein, peptides or polypeptides that are removed from their natural environment, isolated, or separated.
By the term "comprising" it is meant that the isolated polypeptide in accordance with the present disclosure includes the amino acid sequence denoted by SEQ ID NO: 1, as detailed above, but may also include additional amino acid residues at the N-terminus or at the C-terminus of the peptide or at both termini.
Furthermore in various embodiments the isolated polypeptide according to the present disclosure encompasses a polypeptide comprising the amino acid sequence denoted by SEQ ID
NO: 1, in which the N-terminus and/or the C-terminus of the peptide denoted by SEQ ID NO: 1 carries a protecting group. Protective (or protecting) groups are well known in the art and include inter cilia alcohol protecting groups, amine protecting groups, carbonyl protecting groups and others.
As indicated above, the present disclosure also encompasses a functional fragment or derivative of the isolated polypeptide as defined herein.
The term "fragment" as herein defined refers to any peptide or polypeptide which is at least one amino acid shorter than the isolated polypeptide in accordance with the present disclosure, obtained by deletion of at least one amino acid residue from the polypeptide in accordance with the present disclosure.
In some embodiments, a fragment of the isolated polypeptide in accordance with the present disclosure is a polypeptide that comprises a contiguous amino acid portion of SEQ ID NO:
1, that is 1, 2, 3, 4, 5 or more amino acid residues shorter than the sequence denoted by SEQ ID
NO: 1.
By the term "derivative" or "derivatives" it is meant to include polypeptides, which comprise the amino acid sequence denoted by SEQ 11) NO: 1, but differ in one or more amino acid residues in their overall sequence, namely, which have deletions, substitutions (e.g. replacement of at least one amino acid by another amino acid), inversions or additions within the overall sequence. This term also encompasses the replacement of at least one amino acid residue in the overall sequence by its respective L amino acid residue.
Derivatives also encompass amino acid sequence denoted by SEQ ID NO: 1, in which at least one amino acid residue is replaced by a synthetic amino acid residue, an amino acid analogue or an amino acid mimetic.
As known in the art, amino acid "substitutions" are the result of replacing one amino acid with another amino acid, for example with another amino acid that has similar structural and/or chemical properties (conservative amino acid replacements). Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, each of the following eight groups contains amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).
It is appreciated that the fragment or derivative of the isolated polypeptide as defined herein must not alter the biological activity of the original polypeptide. By the term "functional' it is meant to encompass any fragment or derivative of the amino acid sequence denoted by SEQ ID
NO: 1, which retains a biological activity qualitatively similar to that of the unmodified polypeptide (namely a polypeptide having the amino acid sequence denoted by SEQ ID NO: 1.
The biological activity of the fragment or derivative as herein defined may be determined by any method known in the art, for example by monitoring the effect of administering the polypeptide as herein defined to model cells or animals In particular embodiments, the present disclosure relates to a functional fragment or derivative of the isolated polypeptide comprising amino acid sequence denoted by SEQ ID NO. 1, wherein said functional fragment or derivative has an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, more preferably 95%, in particular 96%, 97%, 98% or 99% identical to the amino acid sequence of the unmodified isolated polypeptide of the present disclosure, namely to one of the amino acid sequences denoted by SEQ ID NO: 1.
In specific embodiments, the functional fragment or derivative of the polypeptide as herein defined has at least 70% identity to the amino acid sequence denoted by SEQ ID
NO. 1. In further specific embodiments, the polypeptide as herein defined consists of the amino acid sequence denoted by SEQ ID NO. 1.
It should be appreciated that the terms "identical", or percent "identity", in the context of two or more amino acids or nucleic acids sequences, refer to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70%, identity, 75% identity, preferably 80%, 85%, 90%, or higher identity) over a specified region, when compared and aligned for maximum correspondence over a comparison window.
The peptide of the present disclosure may be administered per se or in combination with another active agent.
Thus in various further embodiments, the method as herein defined further comprises administering at least one additional therapeutic agent to said subject.
By the term "additional therapeutic agent" in the context of the present disclosure it is referred to any therapeutic agent known to a skilled physician for the treatment or prevention of a neurodegenerative disease. For example, Aricept , Exelon , Razadyne , Memantine, Namenda or Levodopa, to name but few, may be used as additional therapeutic agents for the treatment of the respective neurodegenerative disease as further detailed below.
In some embodiments, the subj ect suitable for the methods of the present disclosure is not suffering from any type of cancer. In some specific embodiments, the subject is not suffering from brain cancer, e.g. neuroblastoma.
All the embodiments as described above and further below, concerning the uses, compositions and kits of the isolated peptide according to the present disclosure are relevant to all the different aspects disclosed herein.
In a second of its aspects, the present disclosure further provides a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypepti de or a salt thereof.
In some embodiments the peptide of the present disclosure may be administered in a pharmaceutical composition along with a pharmaceutically acceptable excipient, carrier, or diluent.
Administration of the isolated polypeptide or a functional fragment or derivative thereof or a pharmaceutical composition comprising the same according to the present disclosure may be performed by any one of the acceptable routes, for example but not limited to oral administration, intravenous, intramuscular, intraperitoneal, intrathecal or subcutaneous injection, intra-rectal admini strati on, intranas al administration, ocular administration or topical administration.
As mentioned above, the functional fragment or derivative according to the present disclosure has at least 70% identity to the amino acid sequence denoted by SEQ
ID NO. 1.
In some more specific embodiments, the polypeptide consists of the amino acid sequence denoted by SEQ ID NO. 1.
In a further aspect, the present disclosure provides a combination therapy comprising an isolated peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or a functional derivative thereof or a pharmaceutically acceptable salt of said isolated peptide and at least one additional therapeutic agent for use in a method of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
In another aspect, the present disclosure provides a combination therapy comprising an isolated peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or a functional derivative thereof or a pharmaceutically acceptable salt of said isolated peptide and at least one additional therapeutic agent for use in improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
In some embodiments, the at least one additional therapeutic agent may refer to any therapeutic agent known to a skilled physician for the treatment or prevention of Alzheimer's disease.
Although current medications cannot cure Alzheimer's, some treatments address the underlying biology. Other medications may help lessen symptoms, such as memory loss and confusion. To date, the U.S. Food and Drug Administration (FDA) has approved medications that fall into two categories: drugs that may change disease progression in people living with Alzheimer's, and drugs that may temporarily mitigate some symptoms of Alzheimer's disease.
The drugs that may change disease progression may provide benefits to both cognition and function in people living with Alzheimer's disease For example, Aducanumab (AduhelmTM) is an anti-amyloid antibody intravenous (IV) infusion therapy approved for Alzheimer's disease. In some embodiments, the at least one additional therapeutic agent may be Aducanumab (AduhelmTm).
Among the drugs that treat symptoms, there are some for cognitive symptoms and others for non-cognitive symptoms (memory and thinking).
While medications that treat cognitive symptoms do not stop the damage Alzheimer's causes to brain cells, they may help lessen or stabilize symptoms for a limited time by affecting certain chemicals between the brain's nerve cells.
The following medications are non-limiting examples of drugs prescribed to treat symptoms related to memory and thinking.
In some embodiments, the at least one additional therapeutic agent may be a cholinesterase inhibitor.
Cholinesterase inhibitors are prescribed to treat symptoms related to memory, thinking, language, judgment and other thought processes. These medications prevent the breakdown of acetylcholine, a chemical messenger important for memory and learning. These drugs support communication between nerve cells.
Non-limiting examples of cholinesterase inhibitors suitable according to the present disclosure are: Donepezil (Aricept0) approved to treat all stages of Alzheimer's disease, Rivastigmine (Exelon0) approved for mild-to-moderate Alzheimer's as well as mild-to-moderate dementia associated with Parkinson's disease and Galantamine (Razadyneg) approved for mild-to-moderate stages of Alzheimer's disease.
In some embodiments, the at least one additional therapeutic agent may be a glutamate regulator.
Glutamate regulators are prescribed to improve memory, attention, reason, language and the ability to perform simple tasks. This type of drug works by regulating the activity of glutamate, a different chemical messenger. For example, Memantine (Namenda0) was approved for moderate-to-severe Alzheimer' s disease.
Furthermore, an additional exemplary treatment may be a combination of a cholinesterase inhibitor and a glutamate regulator such as Donepezil and memantine (Namzaric0) approved for moderate-to-severe Alzheimer's disease.
An example of medications for treating non-cognitive symptoms of Alzheimer's disease i.e. behavioral and psychological symptoms such as sleep disturbances, agitation, hallucinations and delusions may be an Orexin receptor antagonist. Orexin receptor antagonist such as Suvorexant (Belsomra0) is prescribed to treat insomnia for individuals living with dementia, this drug is thought to inhibit the activity of orexin, a type of neurotransmitter involved in the sleep-wake cycle and is approved for mild-to-moderate Alzheimer's disease.
In some embodiments, the at least one additional therapeutic agent suitable for the combination therapy disclosed herein is any one of cholinesterase inhibitors, glutamate regulators, a combination of a cholinesterase inhibitor and a glutamate regulators, Orexin receptor antagonist or Aducanumab.
In certain embodiments, the isolated peptide and said additional therapeutic agent are administered concomitantly or consecutively.
Still further in an additional aspect, the present disclosure provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated polypeptide or of a salt thereof.
In some embodiments, the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is wherein the method results in amelioration or reduction of at least one condition or symptom associated with at least one of a neurodegenerative disease and cognitive decline in a subject in need thereof.
In other embodiments, the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is wherein the condition or symptom is at least one of anxiety, short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, impaired glucose metabolism, oxidative stress or any combination thereof.
In further embodiments the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is wherein the method results in improvement of at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof In some embodiments, the isolated polypeptide according to the present disclosure is for use in a method for preventing or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
In further embodiments, the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is for preventing, treating, ameliorating, or delaying the onset of cognitive decline.
As mentioned above, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Dementia, a disorder associated with protein misfolding, cognitive decline, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MIND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Fri edreich's Ataxia, multiple sclerosis, Idiopathic Intracranial Hypertension, Cranial neuropathies, Trigeminal neuralgia, frontotemporal dementias (FTD), Senile Dementia (Dementia NOS), Motor Neuron Disease, or bipolar disorder.
In various embodiments the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is applicable to Alzheimer's disease, Parkinson's disease, Dementia or cognitive decline.
In some embodiments, neurodegenerative disease is Alzheimer's disease.
In certain embodiments, the isolated polypeptide, functional fragment or derivative or salt thereof for use according to the present disclosure is for preventing, treating, ameliorating, or delaying the onset of cognitive decline.
In some embodiments, the functional fragment or derivative of the isolated peptide as disclosed herein has at least 70% identity to the amino acid sequence denoted by SEQ lD NO. 1.
In certain embodiments, the polypeptide consists of the amino acid sequence denoted by SEQ ID NO. 1.
In some further embodiments, the polypeptide is able to penetrate the Blood Brain Barrier.
As mentioned above, the isolated peptide as disclosed herein is for use in a method that results in protection from oxidative stress in the neurons of said subject.
In some specific embodiments, the isolated peptide modulates the expression of at least one gene associated with protection from oxidative stress In certain embodiments, the at least one gene is any one of SOD1, BDNF and TH.
As mentioned above, the isolated peptide as disclosed herein is for use in a method that results in increasing glucose metabolism and/or transport in the neurons of said subject.
In some specific embodiments, the isolated peptide modulates the expression of at least one gene associated with glucose metabolism and/or transport.
In certain embodiments, the at least one gene is any one of GLUT1, GLUT3 and P53.
As mentioned above, the isolated peptide as disclosed herein is for use in a method comprising administering a pharmaceutical composition comprising said isolated polypeptide or a functional fragment or derivative of said isolated peptide or a salt thereof.
In some further embodiments, the isolated peptide as disclosed herein is for use in a method further comprising administering at least one additional therapeutic agent to said subject.
In a further aspect, the present disclosure provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated peptide or a salt thereof for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated peptide or of a salt thereof.
As mentioned above in some embodiments, the functional fragment or derivative of the isolated peptide as disclosed herein has at least 70% identity to the amino acid sequence denoted by SEQ ID NO. 1. In certain embodiments, the polypeptide consists of the amino acid sequence denoted by SEQ ID NO. 1.
Still further the present disclosure provides a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent, for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
In some embodiments, the pharmaceutical composition comprising the isolated peptide as disclosed herein is use in a method for preventing or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
By a further aspect thereof the present disclosure provides a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ
ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent, for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof The term "pharnuzceutical composition" as herein defined generally comprises as an active agent an isolated polypeptide according to the present disclosure (namely an isolated polypeptide comprising or consisting of the amino acid sequence denoted by SEQ
ID NO: 1) or a functional fragment or derivative of the isolated polypeptide or a salt thereof, and at least one of a buffering agent, an agent which adjusts the osmolarity thereof, and optionally, at least one pharmaceutically acceptable excipient, carrier, diluent and/or additive as known in the art.
As used herein the term "pharmaceutically acceptable excipient, carrier, diluent and/or additive" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like, as known in the art. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.
The pharmaceutical compositions according to the invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutically acceptable carrier(s), excipient(s) or additive(s).
For example, the term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents as well known in the art. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
The pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
Supplementary or additive active ingredients, for example additional anti-cancer agents, can also be incorporated into the pharmaceutical composition of the invention.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
By the term "effective amount" as used herein it is meant an amount necessary to achieve a desirable result. The effective amount is determined by the severity and type of the disease or condition in conjunction with the preventive or therapeutic objectives, the route of administration and the patient's general condition (age, sex, weight and other considerations known to the attending physician). The effective amount may be determined based on animal models and is determined by a skilled physician.
By the term "subject in need thereof' or "subject" as used herein it is meant to include warm-blooded animals (such as for example humans, rats, mice, dogs, cats, guinea pigs and primates). In particular embodiments, the subject is human.
In some embodiments the subject according to the present disclosure is diagnosed by a skilled physician as being at a risk of developing a neurodegenerative disease or cognitive decline.
In some other embodiments the subject is diagnosed as having the disease, disorder or condition as herein defined. Diagnosis of the disease, disorder or condition as herein defined may be performed by a skilled physician, by means and steps as known in the art.
In certain embodiments, the polypeptide of the pharmaceutical composition for use according to the present disclosure consists of the amino acid sequence denoted by SEQ ID NO.
1.
In some particular embodiments, the neurodegenerative disorder is Alzheimer's disease In another aspect, the present disclosure further provides a kit comprising:
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof; and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for at least one of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
A further aspect of the present disclosure provides a kit comprising:
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof; and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
The kit according to the present disclosure may further include instructions for use.
The isolated polypeptide according to the present disclosure or a functional fragment or derivative of the isolated polypeptide or a salt thereof and the at least one pharmaceutically acceptable excipient, carrier or diluent may be contained in the same container or in different ones.
Still further, the kit according to the present disclosure may include container means for containing separate kit components. In some embodiments the kit of the present disclosure further includes at least one solvent or buffer known in the art.
The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20%
higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. As used herein the term "about" refers to 10 %.
The terms "comprise", "comprises", "comprising","includes","including", "having" and their conjugates mean "including but not limited to". This term encompasses the term "consisting of". Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises"
and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. The following examples are representative of techniques employed by the Inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.
It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.
Experimental procedures Mice ICR female mice having initial body weight of 20-22 grams were used (Envigo).
Blood brain barrier (BBB) penetration assay A control group of 6 mice was injected intraperitoneally (I.P.) with 5%
mannitol solution and another group of 6 mice was injected I.P. with the polypeptide dTCApFs (also termed herein Nerofe, at 60 mg/kg) in a 5% mannitol solution. Spinal fluid was collected after the injection (1 hour) and subjected to ELISA analysis of dTCApFs (Nerofe), as detailed below.
ELISA analysis of niice cerebrospinal fluids Mice were injected with 60 mg/kg D-amino acid dTCApFs (Nerofe), and cerebrospinal fluid (about 10 pl) and serum were collected after 1 hr, and frozen. A
calibration curve was prepared by serial dilutions of the polypeptide in PBS (Biological industries, 02-023-5A) as noted in table 1 below.
Next, the cerebrospinal samples were thawed and kept on ice, and then diluted in 220 ill PBS. Sample loading was next performed by loading 100 ul duplicates of each sample and of the standard in a Maxisorp 96-wells plate (NUNC, F96 Maxisorp, 442404). The plate was sealed and incubated at 4 C overnight with gentle shaking. The plate was washed by removing the liquid and washing the plate four (4) times using a multi-pipette or an automated washer with 300 41 0.05%
TW-20 (Amresco, 0777-1L) in PBS. The samples were blocked by diluting 5% BSA
(MP
biomedicals, 160069) in PBS. Then, 300 ul of the blocking buffer were loaded in each well and the plate was incubated at room temperature (R.T.) for 1 hour with gentle shaking. The Plates were washed again, as described above.
Detection was performed by diluting the first antibody according to table 1 below in diluent (0.05% TW-20, 0.1% BSA in PBS). Then, 100 ul of detection antibody were loaded into each well and the plate was incubated at R.T. for 2:00 hours with gentle shaking. The plate was washed again as detailed above. Next, FIRP conjugate was prepared by diluting goat anti-rabbit HRP conjugate antibody (Cell signaling, 7074) according to table 1 in diluent. HRP conjugate (100 pi) was then loaded into each well and the plate was incubated for 1:00 hour at R.T. with gentle shaking. The plate was washed again, as detailed above.
Development was performed by adding 100 pl TMB (Milipore, ES001-500ML) to each well, and waiting for blue color development, then adding 100 ul 2N H2SO4 (Frutarom, 5552540).
Plate reading was performed in a microplate reader, by checking the wells absorbance at 450 nm.
Table 1 Calibration curve preparation Peptide Calibration curve 1st antibody dilution Secondary antibody range dilution dTCApFs 1000-15.6 pg/ml 1:250 1:300 Preparation of the dTCApFs peptide and compositions comprising thereof The polypeptide termed herein dTCApFs (or Nerofe, both terms are used herein interchangeably and refer to the same peptide as indicated above) is a 14 amino acid residues long peptide, in which all of the amino acid residues are at their D configuration, having the amino acid sequence of Trp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp Glu Arg Lys (or WWTFFLPSTLWERK
in a single letter code, as denoted by SEQ ID NO: 1). A peptide having an amino acid sequence of Trp Trp Thr Phe Phe Leu Pro Ser Thr Leu Trp Glu Arg Lys (or WWTFFLPSTLWERK in a single letter code, as denoted by SEQ ID NO: 2) in which all of the amino acid residues are at their L
configuration, was also used. The above D-amino acid peptide was GlVIP
manufactured in BCN
peptides at Barcelona. The above L-amino acid peptide was manufactured at Anaspec Inc., in the USA.
Cell viability assay (Resazurin) of cells treated with dTCApFs (Nerofe) Materials:
Clear bottom 96 well cell culture plates (Greiner 60-65590) Cell Culture Media:
10% FBS (Biological industries, cat# 04-121-1A) RPMI (Gibco cat# 21875034) 100 u/ml penicillin and 100 l_tgind streptomycin (Biological Industries) 250 ng/ml Amphotericin B (Biological Industries) Cell Treatment Media: Cell Culture media + 5% Mannitol (Filter after Mannitol addition) Cell Cytotoxicity Assay Kit (Colorimetric): ab112118 abcam Cell line SH-SY5Y (ATCCe CRL-2266Tm) For Treatment:
2 mg/ml dTCApFs (Nerofe) stock solution: Dilute 100 Ill of 20 mg/ml dTCApFs (Nerofe) in 900 pi Cell treatment media 4u8C (sigma SIM-1,0949) Cyclic Pifithrin-cc hydrobromide (sigma P4236) Table 2 Treatment regime 4u8C 4 hours before Cyclic Pifithrin-a dTCApFs 24 mM
H202 24 hours dTCApFs (i mM) hydrobromide hours (mg/ml) 4 hours before dTCApFs (mM) 0 0.1 0.025 5 0 0.5 0.025 5 0 1 0.025 O 10 0.1 0.025 O 10 0.5 0.025 0 10 1 0.025 5 0 0 0.025 O 10 0 0.025 O 0 0 0.025 no cells no cells Procedure The cells were seeded at 5,000 cells/well in a black 96 well plate with clear bottom, in 100 ml growing media. All unused wells were filled with 100 ml media. The cells were incubated for 24 hours. Next, the media was removed, and the cells were first treated with inhibitors (namely 4u8c and Cyclic Pifithrin-ot hydrobromide) in 50 ml treatment media. The cells were then incubated for four (4) hours. The cells were next treated with dTCApFs (Nerofe) by adding 50 ml treatment media with dTCApFs (a x2 dTCApFs stock was prepared and added appropriately to each well). Control wells were filled with 50 ml treatment media. The cells were incubated for 24 hours. Then the media was removed and 100 ml H202 were added to each well in growing media.
Next, 100 ml growing media were added to the control wells. The cells were incubated for 24 hours.
Finally, 20 ml of Cell Cytotoxicity Assay Kit (Colorimetric) ab112118 were added to each well, and the cells were incubated for 4 hours. The cells were read at excitation 530 and emission 590, sensitivity 40, using Biotek Synergy-HT plate reader. The results were calculated as follows:
the percentage of cells viability compared to control was calculated.
Expression of the proteins Ghia, Xl3P1 and p53 in human neuroblastoma cells The expression levels of )(BPI, p53 and Glutl were examined by Western blot of cells treated with dTCApFs (the all D-amino acid residue polypeptide) and the corresponding L-amino acid residue polypeptide.
Materials for cell growth:
- Growing Medium -o DMEM high glucose, L-Glutamine (Gibco 41965-039) o Sodium Pyruvate 11.0 mg/ml (100 mM) (Biological industries cat No. 03-042-1B) o 50 ml FBS (Biological industries cat no. 04-121-1A).
o 0.5 ml Amphotericin B 2500 pg/ml (Biological industries cat no. 03-029-1).
o Penicillin-Streptomycin Solution (Biological Industries cat no. 03-031) - Treatment Medium ¨
o Growing Medium o 5% Mannitol (Sigma cat no. M4125-500G) The media is filtered in 0.2 pm filter after adding Mannitol.
- Trypsin EDTA (Biological industries cat no. 03-052-1B).
- 175cm2 culture Flasks - 20 mg/m1 all D-amino acid residue dTCApFs (Nerofe) or the corresponding all L-amino acid residue polypeptide in Mannitol (aliquots are used, avoid repeated freeze-thaw cycle).
- SIISY5Y cells line Materials for Western blot analysis:
- Ly si s Buffer containing:
o Ripa Buffer (Sigma R0278) o Protease inhibitor (HaltTM Protease Inhibitor Cocktail, PIR-78410) o phosphatase inhibitors (Sigma P5726) o Benzonase Nuclease, Purity > 90% (70746 Millipore) 25U/m1 - X4 LDS sample buffer (invitrogen cat# NP0007) - Sample reducing agent (Invitrogen cat#NP0009, 10-fold concentrated) - 4-20% Tris-Glycin gel (NuSep cat# NG21-420) - 10x TRIS/GLYCINE transfer BUFFER (Bio-Rad 1610734) - PVDF membrane (immobilon-P 0.45um) - TBST buffer: TBS (J640 Amersco) + 0.05% Tween-20 - 5% skim milk in TBST
- l' antibodies diluted in 5% skim milk in TBST
o Anti-KBP1 antibody (ab220783) diluted 1:1,000 o Anti-P53 antibody (ab28) diluted 1:1,000 o Anti-Glutl antibody (ab115730) diluted 1:10,000 - Secondary antibodies diluted 1:10,000 in 5% skim milk in TBST:
o Anti-mouse IgG, HRP-linked Antibody #7076 (Cells signaling) o Anti-rabbit IgG, TARP-linked Antibody #7074 (Cells signaling) - SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Scientific 34095) Cell culture - Passages:
Cells were grown in an incubator at 37 C, 5% CO2 100% humidity until reaching 70-80%
density on the flask (and not more). The flask was emptied using a pipette.
Then Trypsin EDTA
were added and the flask was left in the incubator until most of the cells were observed to be not attached to the flask (while avoiding tapping on the flask to increase detaching of the cells), this procedure takes a few minutes. Then 10 ml medium were added to the Trypsin and the medium and Trypsin were divided into 2 flasks, to which 15 ml fresh medium were added. The cells were left to grow 2-3 days until reaching 70-80% and the passage procedure was repeated.
Cells treatment:
The cells were grown until reaching 70-80% density on the flask (and not more). The cells were detached from the flask by following the Trypsin EDTA procedure detailed above. The cells in medium and Trypsin were transferred into 50 ml conical tube and centrifuged at 300g for 10 min in 4 C. The supernatant was discarded and 15m1 fresh medium were added to the cells pellet, fluidizing the pellet. Cells were then counted, seeded at 1.5 M cells/75 CM2 flask in 30 ml growing media. The cells were incubated overnight The following day, cell treatment medium was prepared as follows (medium including 5%
mannitol):
= Control = 20 jig/ml all D-amino acid residue dTCApFs (SEQ ID NO: 1) = 30 lug/m1 all D-amino acid residue dTCApFs (SEQ ID NO: 1) = 40 jig/ml all D-amino acid residue dTCApFs (SEQ ID NO: 1) = 50 jig/ml all D-amino acid residue dTCApFs (SEQ ID NO: 1) = 20 lAg/m1 all L-amino acid residue polypeptide corresponding to dTCApFs (SEQ ID
NO: 2) = 30 pg/m1 all L-amino acid residue polypeptide corresponding to dTCApFs (SEQ ID
NO: 2) = 40 pg/m1 all L-amino acid residue polypeptide corresponding to dTCApFs (acid residue polypeptide corresponding to dTCApFs) = 50 pug/m1 all L-amino acid residue polypeptide corresponding to dTCApFs (SEQ ID
NO: 2) The media was aspirated from the flasks, and 30 ml of each treatment was added to the corresponding flask. The cells were then incubated for 48 hours and then collected for Western Blot analysis, as detailed below.
Western blot analysis procedure:
Media was discarded and the cells were washed twice with PBS. PBS (5 ml) was added to each flask, the cells were scraped with a cell scraper, and collected into a 15 ml conical tube, which was centrifuged 10 minutes at 300G, 4 C. The supernatant was then discarded and 90-200 ml lysis buffer was added to the cell pellet, the mixture was pipetted up and down, and incubated 20 minutes on ice. The lysed cells were then centrifuged 15K G for 15 minutes at 4 C, and the supernatant was collected.
Reducing agent was added to each sample, then running buffer (x4 LDS) was added to each sample and the sample was boiled for 5 minutes, vortexed, centrifuged (for Glutl, the sample was not boiled). Next, 30-50 ml of the sample was loaded onto 4-20% Tris Glycin gel and the gel was transferred using semi-dry wet transfer device with rapid transfer buffer onto PVDF
membrane 20-60 minutes at 20V. The membrane was blocked for 1 hour with 5%
skim milk in TBST with shaking. Then the first antibody was added, for an over-night incubation period at 4 C
with shaking. The membrane was washed three (3) times with TBST and a secondary antibody was added, for an incubation period of 1 hour at room temperature (R.T.) with shaking.
Development was performed with an ECL substrate, 5 minutes at R.T. with shaking and the reading was performed using a Lycor C-Digit digital reader.
Blood brain barrier (BBB) penetration ability of dTCApFs in mice In order to study the effect of the polypeptide dTCApFs (having the amino acid sequence denoted by SEQ ID NO: 1, also termed herein Nerofe) on brain function, the blood brain barrier (BBB) penetration ability of the polypeptide was examined in mice.
Therefore, a control group of mice was injected intraperitoneally (IP.) with 5% mannitol solution and another group of mice was injected I.P. with the polypeptide dTCApFs (at 60 mg/kg) in 5% mannitol solution and the spinal fluid of the tested mice was then collected and subjected to analysis, as detailed above.
As shown in Figure 1A, right bar, the level of dTCApFs in mice brain was dramatically increased following dTCApFs injection, as compared to the level of dTCApFs in the control group (mice injected with 5% mannitol solution, left bar).
The above results show that dTCApFs has a good penetration ability of the BBB
in mice.
Further to the above experiment and in comparison thereto, the above experiment was peformed by injection to mice of a polypeptide having the same amino acid sequence as that of dTCApFs, in which all of the amino acid residues are in their L configuration (as denoted herein by SEQ ID NO: 2). The results presented in Figure 1B show that this polypeptide is naturally present in mice brain (as judged by the level of the polypeptide in the left bar of Figure 1B) and therefore the level thereof was changed insignificantly upon injection.
The effect of dTCApFs on human neurons in the presence of oxidative stress SH-SY5Y is a human derived cell line first isolated from a bone marrow biopsy taken from a four-year-old female with neuroblastoma. SH-SY5Y cells are often used as an in vitro model of neuronal function and differentiation, for example to study Parkinson's disease and other characteristics of brain cells.
Using human SH-S Y5 Y cells model, dTCApFs was shown to protect human neurons from hydrogen peroxide (H202) insults, in a dose dependent manner, as detailed below.
To this end, SH-SY5Y cells were treated with H202 in absence or in the presence of increasing doses of dTCApFs (at 0, 0.5, 1, 1.5 and 2 jig/m1) for 24 hours.
Cell viability was measured using the Resazurin reagent, as detailed above. The upper graph in Figure 2 relates to SH-SY5Y cells treated with 0.01 uM H202 in the presence of increasing doses of dTCApFs. The lower graph in Figure 2 relates to SH-SY5Y cells treated with a higher concentration of H202 (0.1 uM) in presence of increasing doses of dTCApFs. As can be seen in Figure 2, at the absence of dTCApFs, the level of cell viability was low in cells treated with two different doses of H202 (namely about 65% in cells treated with 0.1 uM H202 and about 75% in cells treated with 0.01 uM
H202). Surprisingly, increasing doses of dTCApFs increased viability of cells in a dose depended manner, in both tested doses of H202.
The effect of dTCApFs on gene expression in human neurons (SH-SY5Y cells) Without wishing to be bound by theory, the above results may indicate that dTCApFs has a protective effect on human neurons from oxidative stress, by inducing the expression of SOD1.
Therefore, the mechanism by which SH-SY5Y cells become resistant to oxidative stress in the presence of dTCApFs was next examined.
Using reverse-transcription polymerase chain reaction (RT-PCR), the levels of different genes that may be associated with protection against oxidative stress were determined. Among the genes tested were SOD1, Catalase and others, as detailed below.
SOD1 (Superoxide dismutase also known as superoxide dismutase 1) is an enzyme that in humans is encoded by the SOD1 gene. SOD1 is implicated in apoptosis and familial amyotrophic lateral sclerosis. As known in the art, SOD1 is reactive oxygen species release during oxidative stress.
The gene expression level assay was performed as detailed above. Briefly, SH-SY5Y cells were incubated in the presence of increasing doses of dTCApFs (namely 0, 0.5, 1, 1.5 and 2 jig/ml) and after 24 hours of incubation, cells were harvested and subjected to RT-PCR
analysis, directed to different genes, among which were: human beta-actin, BDNF (brain-derived neurotrophic factor, middle graph in Figure 3), SOD1 (Superoxide dismutase, upper graph in Figure 3), CATALASE (not shown) and tyrosine hydroxylase (TH, bottom graph in Figure 3).
The induction of the different genes was calibrated against the level of expression of human beta-actin.
BDNF (brain-derived neurotrophic factor, or abrineurin) is a protein that in humans, is encoded by the BDNF gene. BDNF is a member of the neurotrophin family of growth factors, which are related to the canonical nerve growth factor. Neurotrophic factors are found in the brain and the periphery.
As shown in Figure 3, SOD1 was strongly induced by dTCApFs, in a dose dependent manner. BDNF and TH were slightly induced, at some dTCApFs levels. These results indicate that dTCApFs induced a protective effect to the cells, for example via up regulating the expression of SOD1, which assists in cell protection against oxidative stress which is one of the manifestations of the development of neurodegenerative diseases and disorders.
The effect of dTCApFs on glucose transporters in human neurons Similar to the other organs in the body, the brain is dependent on glucose as an energy source and requires the expression of glucose transporter proteins to enable passage of glucose across the blood-brain barrier and the plasma membranes of nerve cells. At least two isoforms of glucose transporters which participate in neuronal glucose transport, are known, namely GLUT1 and GLUT3.
In order to study the effect of the polypeptide dTCApFs on the expression level of the above glucose transporters (GLUT1 and GLUT3), SH-SY5Y cells were incubated in the presence of increasing doses of dTCApFs (namely 1, 5 and 10 ttg/m1) and after 24 hours of incubation, the cells were harvested, and equal amounts of total protein amounts of the different samples were subjected to Western blot analysis. The expression level of various proteins was monitored, among which were human beta-actin, Glutl and Glut3. As shown in Figure 4, the levels of Glutl and Glu3 proteins were increased with the increase in dTCApFs doses.
It has been previously postulated that oxidative damage is among the key components to the etiology of neurodegenerative diseases. In various neurodegenerative conditions, for example Alzheimer's disease and amnestic mild cognitive impairment, glucose metabolism is dramatically decreased, probably owing, at least in part, to oxidative damage to enzymes involved in glucose metabolism. Consequently, processes required for cognitive function are impaired, and synaptic dysfunction and neuronal death result.
In view of the above results, which demonstrate that incubation of SH-SY5Y
cells in the presence of dTCApFs induced expression of the Glutl and Glut3 proteins, a further beneficial protective effect of the polypeptide on brain function is suggested.
Without wishing to be bound by theory, the effect of dTCApFs observed above suggests that a protective and therapeutic effect on the brain via lowering oxidative stress and positively affecting glucose metabolism.
In other words, in-vitro studies performed with human SH-SY5Y cells shows that the polypeptide dTCApFs protects cells from oxidative stress and increases glucose uptake.
Furthermore, in-vitro experiments performed with human microglial cells show that dTCApFs (Nerofe) can downregulate secretion levels of proinflammatory cytokines, such as IL-6, TNFcc and soluble ST2 (data not shown).
Comparing the effect of dTCApFs and a polypeptide having the same amino acid sequence in all L configuration on protein expression in human neurons As detailed above, human neuroblastoma cells (SH-SY5Y cells), are considered to be an acceptable in vitro model for Alzheimer's disease (AD). In order to examine the effect on these cells of the dTCApFs polypeptide (having the amino acid sequence denoted herein by SEQ ID
NO: 1) and in parallel of the polypeptide having the amino acid sequence of dTCApFs in the all L
amino acid configuration (having the amino acid sequence denoted herein by SEQ
ID NO: 2), SH-SY5Y cells were incubated in the presence of each one the above polypeptide, as detailed above.
Specifically, the effect of the above polypeptides on the expression levels of the proteins Glucose Transporter 1 (GLUT1), X-Box Binding Protein 1(XBP1) and Tumor protein P53 (P53) in these cells was tested.
As detailed above, the protein Glutl is an important glucose transporter in the human brain.
XBP1 (X-box binding protein 1) in a very important transcription factor in human brain that is involved with increasing memory and cognitive capabilities and p53 in a negative regulator of glucose metabolism in the brain and is highly expressed in Synculin plaques.
As demonstrated in Figure 5C, the all D-amino acid dTCApFs (Nerofe) polypeptide was highly efficient in increasing the expression level of the protein Glutl (about 3-9 fold, as shown in Figure 5C), as opposed to almost no effect on the expression level of this protein in the presence of the L-amino acid polypeptide (shown in Figure 6C).
As detailed above, XBP1 is an important transcription factor in human brain to which a beneficial effect on memory and cognitive capabilities is attributed. As shown in Figure 5A, expression of the protein )(BPI was highly induced in the presence of the dTCApFs polypeptide (for example about 6-fold in the presence of 50 jig/ml dTCApFs and up to 14-fold in the presence of 20 jig/ml dTCApFs as opposed to a rather mild effect on the expression level of this protein in the presence of the L-amino acid polypeptide (about 2-fold in the presence of 20 or 50 itg/ml polypeptide, as shown in Figure 6A).
Furthermore, incubation of the above cells in the presence of the dTCApFs polypeptide completely downregulated p53 expression in these cells, in a concentration dependent manner (Figure 5B) as opposed to a modest down regulation observed in the presence of the all L-amino acid polypeptide (Figure 6B). Lowering the expression level of p53 is considered to be a beneficial effect, since it leads to an increase in glucose metabolism Comparing the effect of dTCApFs and a polypeptide having the same amino acid sequence in all L configuration in the presence of oxidative stress Using human SH-SY5Y cells model, dTCApFs was shown to protect human neurons from hydrogen peroxide (H202) insults better than L-amino acid CTApFs, in a dose dependent manner, as detailed below.
To this end, SH-SY5Y cells were treated with H202 in absence or in the presence of increasing doses of dTCApFs or L-amino acid CTApFs (at 0,0.5, 1, 1.5 and 2 itg/m1) for 24 hours.
Cell viability was measured using the Resazurin reagent, as detailed above.
The graph in Figure 7 relates to SH-SY5Y cells treated with 0.1 mM H202 in the presence of increasing doses of dTCApFs or L-amino acid CTApFs As can be seen in Figure 7, when using L-amino acid CTApFs, the level of cell viability was lower than in cells treated with dTCApFs.
These results provide a further example for a beneficial protective and therapeutic cognitive effect for the dTCApFs polypeptide, via the involvement of the affected proteins in various cellular pathways relating thereto.
2 mg/ml dTCApFs (Nerofe) stock solution: Dilute 100 Ill of 20 mg/ml dTCApFs (Nerofe) in 900 pi Cell treatment media 4u8C (sigma SIM-1,0949) Cyclic Pifithrin-cc hydrobromide (sigma P4236) Table 2 Treatment regime 4u8C 4 hours before Cyclic Pifithrin-a dTCApFs 24 mM
H202 24 hours dTCApFs (i mM) hydrobromide hours (mg/ml) 4 hours before dTCApFs (mM) 0 0.1 0.025 5 0 0.5 0.025 5 0 1 0.025 O 10 0.1 0.025 O 10 0.5 0.025 0 10 1 0.025 5 0 0 0.025 O 10 0 0.025 O 0 0 0.025 no cells no cells Procedure The cells were seeded at 5,000 cells/well in a black 96 well plate with clear bottom, in 100 ml growing media. All unused wells were filled with 100 ml media. The cells were incubated for 24 hours. Next, the media was removed, and the cells were first treated with inhibitors (namely 4u8c and Cyclic Pifithrin-ot hydrobromide) in 50 ml treatment media. The cells were then incubated for four (4) hours. The cells were next treated with dTCApFs (Nerofe) by adding 50 ml treatment media with dTCApFs (a x2 dTCApFs stock was prepared and added appropriately to each well). Control wells were filled with 50 ml treatment media. The cells were incubated for 24 hours. Then the media was removed and 100 ml H202 were added to each well in growing media.
Next, 100 ml growing media were added to the control wells. The cells were incubated for 24 hours.
Finally, 20 ml of Cell Cytotoxicity Assay Kit (Colorimetric) ab112118 were added to each well, and the cells were incubated for 4 hours. The cells were read at excitation 530 and emission 590, sensitivity 40, using Biotek Synergy-HT plate reader. The results were calculated as follows:
the percentage of cells viability compared to control was calculated.
Expression of the proteins Ghia, Xl3P1 and p53 in human neuroblastoma cells The expression levels of )(BPI, p53 and Glutl were examined by Western blot of cells treated with dTCApFs (the all D-amino acid residue polypeptide) and the corresponding L-amino acid residue polypeptide.
Materials for cell growth:
- Growing Medium -o DMEM high glucose, L-Glutamine (Gibco 41965-039) o Sodium Pyruvate 11.0 mg/ml (100 mM) (Biological industries cat No. 03-042-1B) o 50 ml FBS (Biological industries cat no. 04-121-1A).
o 0.5 ml Amphotericin B 2500 pg/ml (Biological industries cat no. 03-029-1).
o Penicillin-Streptomycin Solution (Biological Industries cat no. 03-031) - Treatment Medium ¨
o Growing Medium o 5% Mannitol (Sigma cat no. M4125-500G) The media is filtered in 0.2 pm filter after adding Mannitol.
- Trypsin EDTA (Biological industries cat no. 03-052-1B).
- 175cm2 culture Flasks - 20 mg/m1 all D-amino acid residue dTCApFs (Nerofe) or the corresponding all L-amino acid residue polypeptide in Mannitol (aliquots are used, avoid repeated freeze-thaw cycle).
- SIISY5Y cells line Materials for Western blot analysis:
- Ly si s Buffer containing:
o Ripa Buffer (Sigma R0278) o Protease inhibitor (HaltTM Protease Inhibitor Cocktail, PIR-78410) o phosphatase inhibitors (Sigma P5726) o Benzonase Nuclease, Purity > 90% (70746 Millipore) 25U/m1 - X4 LDS sample buffer (invitrogen cat# NP0007) - Sample reducing agent (Invitrogen cat#NP0009, 10-fold concentrated) - 4-20% Tris-Glycin gel (NuSep cat# NG21-420) - 10x TRIS/GLYCINE transfer BUFFER (Bio-Rad 1610734) - PVDF membrane (immobilon-P 0.45um) - TBST buffer: TBS (J640 Amersco) + 0.05% Tween-20 - 5% skim milk in TBST
- l' antibodies diluted in 5% skim milk in TBST
o Anti-KBP1 antibody (ab220783) diluted 1:1,000 o Anti-P53 antibody (ab28) diluted 1:1,000 o Anti-Glutl antibody (ab115730) diluted 1:10,000 - Secondary antibodies diluted 1:10,000 in 5% skim milk in TBST:
o Anti-mouse IgG, HRP-linked Antibody #7076 (Cells signaling) o Anti-rabbit IgG, TARP-linked Antibody #7074 (Cells signaling) - SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Scientific 34095) Cell culture - Passages:
Cells were grown in an incubator at 37 C, 5% CO2 100% humidity until reaching 70-80%
density on the flask (and not more). The flask was emptied using a pipette.
Then Trypsin EDTA
were added and the flask was left in the incubator until most of the cells were observed to be not attached to the flask (while avoiding tapping on the flask to increase detaching of the cells), this procedure takes a few minutes. Then 10 ml medium were added to the Trypsin and the medium and Trypsin were divided into 2 flasks, to which 15 ml fresh medium were added. The cells were left to grow 2-3 days until reaching 70-80% and the passage procedure was repeated.
Cells treatment:
The cells were grown until reaching 70-80% density on the flask (and not more). The cells were detached from the flask by following the Trypsin EDTA procedure detailed above. The cells in medium and Trypsin were transferred into 50 ml conical tube and centrifuged at 300g for 10 min in 4 C. The supernatant was discarded and 15m1 fresh medium were added to the cells pellet, fluidizing the pellet. Cells were then counted, seeded at 1.5 M cells/75 CM2 flask in 30 ml growing media. The cells were incubated overnight The following day, cell treatment medium was prepared as follows (medium including 5%
mannitol):
= Control = 20 jig/ml all D-amino acid residue dTCApFs (SEQ ID NO: 1) = 30 lug/m1 all D-amino acid residue dTCApFs (SEQ ID NO: 1) = 40 jig/ml all D-amino acid residue dTCApFs (SEQ ID NO: 1) = 50 jig/ml all D-amino acid residue dTCApFs (SEQ ID NO: 1) = 20 lAg/m1 all L-amino acid residue polypeptide corresponding to dTCApFs (SEQ ID
NO: 2) = 30 pg/m1 all L-amino acid residue polypeptide corresponding to dTCApFs (SEQ ID
NO: 2) = 40 pg/m1 all L-amino acid residue polypeptide corresponding to dTCApFs (acid residue polypeptide corresponding to dTCApFs) = 50 pug/m1 all L-amino acid residue polypeptide corresponding to dTCApFs (SEQ ID
NO: 2) The media was aspirated from the flasks, and 30 ml of each treatment was added to the corresponding flask. The cells were then incubated for 48 hours and then collected for Western Blot analysis, as detailed below.
Western blot analysis procedure:
Media was discarded and the cells were washed twice with PBS. PBS (5 ml) was added to each flask, the cells were scraped with a cell scraper, and collected into a 15 ml conical tube, which was centrifuged 10 minutes at 300G, 4 C. The supernatant was then discarded and 90-200 ml lysis buffer was added to the cell pellet, the mixture was pipetted up and down, and incubated 20 minutes on ice. The lysed cells were then centrifuged 15K G for 15 minutes at 4 C, and the supernatant was collected.
Reducing agent was added to each sample, then running buffer (x4 LDS) was added to each sample and the sample was boiled for 5 minutes, vortexed, centrifuged (for Glutl, the sample was not boiled). Next, 30-50 ml of the sample was loaded onto 4-20% Tris Glycin gel and the gel was transferred using semi-dry wet transfer device with rapid transfer buffer onto PVDF
membrane 20-60 minutes at 20V. The membrane was blocked for 1 hour with 5%
skim milk in TBST with shaking. Then the first antibody was added, for an over-night incubation period at 4 C
with shaking. The membrane was washed three (3) times with TBST and a secondary antibody was added, for an incubation period of 1 hour at room temperature (R.T.) with shaking.
Development was performed with an ECL substrate, 5 minutes at R.T. with shaking and the reading was performed using a Lycor C-Digit digital reader.
Blood brain barrier (BBB) penetration ability of dTCApFs in mice In order to study the effect of the polypeptide dTCApFs (having the amino acid sequence denoted by SEQ ID NO: 1, also termed herein Nerofe) on brain function, the blood brain barrier (BBB) penetration ability of the polypeptide was examined in mice.
Therefore, a control group of mice was injected intraperitoneally (IP.) with 5% mannitol solution and another group of mice was injected I.P. with the polypeptide dTCApFs (at 60 mg/kg) in 5% mannitol solution and the spinal fluid of the tested mice was then collected and subjected to analysis, as detailed above.
As shown in Figure 1A, right bar, the level of dTCApFs in mice brain was dramatically increased following dTCApFs injection, as compared to the level of dTCApFs in the control group (mice injected with 5% mannitol solution, left bar).
The above results show that dTCApFs has a good penetration ability of the BBB
in mice.
Further to the above experiment and in comparison thereto, the above experiment was peformed by injection to mice of a polypeptide having the same amino acid sequence as that of dTCApFs, in which all of the amino acid residues are in their L configuration (as denoted herein by SEQ ID NO: 2). The results presented in Figure 1B show that this polypeptide is naturally present in mice brain (as judged by the level of the polypeptide in the left bar of Figure 1B) and therefore the level thereof was changed insignificantly upon injection.
The effect of dTCApFs on human neurons in the presence of oxidative stress SH-SY5Y is a human derived cell line first isolated from a bone marrow biopsy taken from a four-year-old female with neuroblastoma. SH-SY5Y cells are often used as an in vitro model of neuronal function and differentiation, for example to study Parkinson's disease and other characteristics of brain cells.
Using human SH-S Y5 Y cells model, dTCApFs was shown to protect human neurons from hydrogen peroxide (H202) insults, in a dose dependent manner, as detailed below.
To this end, SH-SY5Y cells were treated with H202 in absence or in the presence of increasing doses of dTCApFs (at 0, 0.5, 1, 1.5 and 2 jig/m1) for 24 hours.
Cell viability was measured using the Resazurin reagent, as detailed above. The upper graph in Figure 2 relates to SH-SY5Y cells treated with 0.01 uM H202 in the presence of increasing doses of dTCApFs. The lower graph in Figure 2 relates to SH-SY5Y cells treated with a higher concentration of H202 (0.1 uM) in presence of increasing doses of dTCApFs. As can be seen in Figure 2, at the absence of dTCApFs, the level of cell viability was low in cells treated with two different doses of H202 (namely about 65% in cells treated with 0.1 uM H202 and about 75% in cells treated with 0.01 uM
H202). Surprisingly, increasing doses of dTCApFs increased viability of cells in a dose depended manner, in both tested doses of H202.
The effect of dTCApFs on gene expression in human neurons (SH-SY5Y cells) Without wishing to be bound by theory, the above results may indicate that dTCApFs has a protective effect on human neurons from oxidative stress, by inducing the expression of SOD1.
Therefore, the mechanism by which SH-SY5Y cells become resistant to oxidative stress in the presence of dTCApFs was next examined.
Using reverse-transcription polymerase chain reaction (RT-PCR), the levels of different genes that may be associated with protection against oxidative stress were determined. Among the genes tested were SOD1, Catalase and others, as detailed below.
SOD1 (Superoxide dismutase also known as superoxide dismutase 1) is an enzyme that in humans is encoded by the SOD1 gene. SOD1 is implicated in apoptosis and familial amyotrophic lateral sclerosis. As known in the art, SOD1 is reactive oxygen species release during oxidative stress.
The gene expression level assay was performed as detailed above. Briefly, SH-SY5Y cells were incubated in the presence of increasing doses of dTCApFs (namely 0, 0.5, 1, 1.5 and 2 jig/ml) and after 24 hours of incubation, cells were harvested and subjected to RT-PCR
analysis, directed to different genes, among which were: human beta-actin, BDNF (brain-derived neurotrophic factor, middle graph in Figure 3), SOD1 (Superoxide dismutase, upper graph in Figure 3), CATALASE (not shown) and tyrosine hydroxylase (TH, bottom graph in Figure 3).
The induction of the different genes was calibrated against the level of expression of human beta-actin.
BDNF (brain-derived neurotrophic factor, or abrineurin) is a protein that in humans, is encoded by the BDNF gene. BDNF is a member of the neurotrophin family of growth factors, which are related to the canonical nerve growth factor. Neurotrophic factors are found in the brain and the periphery.
As shown in Figure 3, SOD1 was strongly induced by dTCApFs, in a dose dependent manner. BDNF and TH were slightly induced, at some dTCApFs levels. These results indicate that dTCApFs induced a protective effect to the cells, for example via up regulating the expression of SOD1, which assists in cell protection against oxidative stress which is one of the manifestations of the development of neurodegenerative diseases and disorders.
The effect of dTCApFs on glucose transporters in human neurons Similar to the other organs in the body, the brain is dependent on glucose as an energy source and requires the expression of glucose transporter proteins to enable passage of glucose across the blood-brain barrier and the plasma membranes of nerve cells. At least two isoforms of glucose transporters which participate in neuronal glucose transport, are known, namely GLUT1 and GLUT3.
In order to study the effect of the polypeptide dTCApFs on the expression level of the above glucose transporters (GLUT1 and GLUT3), SH-SY5Y cells were incubated in the presence of increasing doses of dTCApFs (namely 1, 5 and 10 ttg/m1) and after 24 hours of incubation, the cells were harvested, and equal amounts of total protein amounts of the different samples were subjected to Western blot analysis. The expression level of various proteins was monitored, among which were human beta-actin, Glutl and Glut3. As shown in Figure 4, the levels of Glutl and Glu3 proteins were increased with the increase in dTCApFs doses.
It has been previously postulated that oxidative damage is among the key components to the etiology of neurodegenerative diseases. In various neurodegenerative conditions, for example Alzheimer's disease and amnestic mild cognitive impairment, glucose metabolism is dramatically decreased, probably owing, at least in part, to oxidative damage to enzymes involved in glucose metabolism. Consequently, processes required for cognitive function are impaired, and synaptic dysfunction and neuronal death result.
In view of the above results, which demonstrate that incubation of SH-SY5Y
cells in the presence of dTCApFs induced expression of the Glutl and Glut3 proteins, a further beneficial protective effect of the polypeptide on brain function is suggested.
Without wishing to be bound by theory, the effect of dTCApFs observed above suggests that a protective and therapeutic effect on the brain via lowering oxidative stress and positively affecting glucose metabolism.
In other words, in-vitro studies performed with human SH-SY5Y cells shows that the polypeptide dTCApFs protects cells from oxidative stress and increases glucose uptake.
Furthermore, in-vitro experiments performed with human microglial cells show that dTCApFs (Nerofe) can downregulate secretion levels of proinflammatory cytokines, such as IL-6, TNFcc and soluble ST2 (data not shown).
Comparing the effect of dTCApFs and a polypeptide having the same amino acid sequence in all L configuration on protein expression in human neurons As detailed above, human neuroblastoma cells (SH-SY5Y cells), are considered to be an acceptable in vitro model for Alzheimer's disease (AD). In order to examine the effect on these cells of the dTCApFs polypeptide (having the amino acid sequence denoted herein by SEQ ID
NO: 1) and in parallel of the polypeptide having the amino acid sequence of dTCApFs in the all L
amino acid configuration (having the amino acid sequence denoted herein by SEQ
ID NO: 2), SH-SY5Y cells were incubated in the presence of each one the above polypeptide, as detailed above.
Specifically, the effect of the above polypeptides on the expression levels of the proteins Glucose Transporter 1 (GLUT1), X-Box Binding Protein 1(XBP1) and Tumor protein P53 (P53) in these cells was tested.
As detailed above, the protein Glutl is an important glucose transporter in the human brain.
XBP1 (X-box binding protein 1) in a very important transcription factor in human brain that is involved with increasing memory and cognitive capabilities and p53 in a negative regulator of glucose metabolism in the brain and is highly expressed in Synculin plaques.
As demonstrated in Figure 5C, the all D-amino acid dTCApFs (Nerofe) polypeptide was highly efficient in increasing the expression level of the protein Glutl (about 3-9 fold, as shown in Figure 5C), as opposed to almost no effect on the expression level of this protein in the presence of the L-amino acid polypeptide (shown in Figure 6C).
As detailed above, XBP1 is an important transcription factor in human brain to which a beneficial effect on memory and cognitive capabilities is attributed. As shown in Figure 5A, expression of the protein )(BPI was highly induced in the presence of the dTCApFs polypeptide (for example about 6-fold in the presence of 50 jig/ml dTCApFs and up to 14-fold in the presence of 20 jig/ml dTCApFs as opposed to a rather mild effect on the expression level of this protein in the presence of the L-amino acid polypeptide (about 2-fold in the presence of 20 or 50 itg/ml polypeptide, as shown in Figure 6A).
Furthermore, incubation of the above cells in the presence of the dTCApFs polypeptide completely downregulated p53 expression in these cells, in a concentration dependent manner (Figure 5B) as opposed to a modest down regulation observed in the presence of the all L-amino acid polypeptide (Figure 6B). Lowering the expression level of p53 is considered to be a beneficial effect, since it leads to an increase in glucose metabolism Comparing the effect of dTCApFs and a polypeptide having the same amino acid sequence in all L configuration in the presence of oxidative stress Using human SH-SY5Y cells model, dTCApFs was shown to protect human neurons from hydrogen peroxide (H202) insults better than L-amino acid CTApFs, in a dose dependent manner, as detailed below.
To this end, SH-SY5Y cells were treated with H202 in absence or in the presence of increasing doses of dTCApFs or L-amino acid CTApFs (at 0,0.5, 1, 1.5 and 2 itg/m1) for 24 hours.
Cell viability was measured using the Resazurin reagent, as detailed above.
The graph in Figure 7 relates to SH-SY5Y cells treated with 0.1 mM H202 in the presence of increasing doses of dTCApFs or L-amino acid CTApFs As can be seen in Figure 7, when using L-amino acid CTApFs, the level of cell viability was lower than in cells treated with dTCApFs.
These results provide a further example for a beneficial protective and therapeutic cognitive effect for the dTCApFs polypeptide, via the involvement of the affected proteins in various cellular pathways relating thereto.
Claims (52)
1. A method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof.
2. The method according to claim 1, wherein said method results in amelioration or reduction of at least one condition or symptom associated with at least one of a neurodegenerative disease and cognitive decline in a subject in need thereof
3. The method according to claim 2, wherein said condition or symptom is at least one of anxiety, short term memory impairment, long term_ memory impairment, impaired cognitive function, impaired learning function, impaired glucose metabolism, oxidative stress or any combination thereof.
4. The method according to any one of claims 1 to 3, wherein said method results in improvement of at least one of cognitive function, short term memory, long term memory, learning function and glucose metabolism and glucose transport in a subject in need thereof.
5. The method according to any one of claims 1 to 4, wherein said neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Dementia, a disorder associated with protein misfolding, cognitive decline, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia, multiple sclerosis, Idiopathic Intracranial Hypertension, Cranial neuropathies, Trigeminal neuralgia, frontotemporal dementias (FTD), Senile Dementia (Dementia NOS), Motor Neuron Disease, or bipolar disorder.
6. The method according to any one of claims I to 5, wherein said neurodegenerative disease is Alzheimer's disease.
7. The method according to any one of claims 1 to 4, for preventing, treating, ameliorating, or delaying the onset of cognitive decline.
8. The method according to any one of claims 1 to 7, wherein said functional fragment or derivative has at least 70% identity to the amino acid sequence denoted by SEQ
ID NO. 1.
ID NO. 1.
9. The method according to any one of claims 1 to 7, wherein said polypepti de consists of the amino acid sequence denoted by SEQ ID NO. 1 .
10. The method of any one of claims 1 to 9, wherein said polypeptide is able to penetrate the Blood Brain Barrier.
11. The method of any one of claims 1 to 10, wherein said method results in protection from oxidative stress in the neurons of said subject.
12. The method of claim 11, wherein said peptide modulates the expression of at least one gene associated with protection from oxidative stress.
13. The method of claim 12, wherein said at least one gene is any one of Superoxide dismutase 1 (SOD1), Brain-Derived Neurotrophic Factor (BDNF) and Tyrosine Hydroxylase (TH).
14. The method of any one of claims 1 to 13, wherein said method results in increasing glucose metabolism and/or transport in the neurons of said subject.
15. The method of claim 14, wherein peptide modulates the expression of at least one gene associated with glucose metabolism and/or transport.
16. The method of claim 15, wherein said at least one gene is any one of Glucose Transporter 1 (GLUT1), Glucose Transporter 3 (GLUT3) and Tumor protein P53 (P53).
17. The method according to any one of claims 1 to 16, wherein said method comprises administering a pharmaceutical composition comprising said isolated polypeptide or a functional fragment or derivative of said isolated polypeptide or a salt thereof.
18. The method according to any one of claims 1 to 17, wherein said method further comprises administering at least one additional therapeutic agent to said subject.
19. A method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, comprising administering to said subject an effective amount of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof
20. The method according to claim 19, wherein said functional fragment or derivative has at least 70% identity to the amino acid sequence denoted by SEQ ID NO. 1.
21. The method according to claim 19 or claim 20, wherein said polypeptide consists of the amino acid sequence denoted by SEQ ID NO 1
22. A combination therapy comprising an isolated peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or a functional derivative thereof or a pharmaceutically acceptable salt of said isolated peptide and at least one additional therapeutic agent for use in a method of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof
23. A combination therapy comprising an isolated peptide comprising the amino acid sequence denoted by SEQ ID NO. 1 or a functional derivative thereof or a pharmaceutically acceptable salt of said isolated peptide and at least one additional therapeutic agent for use in irnproving at least one of cognitive function, short term memory, long term memoly, learning function, glucose metabolism and glucose transport in a subject in need thereof
24. The combination therapy for use according to claim 22 or claim 23, wherein said at least one additional therapeutic agent is any one of cholinesterase inhibitors, glutamate regulators, a combination of a cholinesterase inhibitor and a glutamate regulators, Orexin receptor antagonist or Aducanumab
25. The combination therapy for use according to any one of claims 22 to 24, wherein said isolated peptide and said additional therapeutic agent are administered concomitantly or consecutively.
26. An isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated polypeptide or of a salt thereof
NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated polypeptide or of a salt thereof
27. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to claim 26, wherein said method results in amelioration or reduction of at least one condition or symptom associated with at least one of a neurodegenerative disease and cognitive decline in a subject in need thereof.
28. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to claim 27, wherein said condition or symptom is at least one of anxiety, short term memory impairment, long term memory impairment, impaired cognitive function, impaired learning function, impaired glucose metabolism, oxidative stress or any combination thereof
29. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 28, wherein said method results in improvement of at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof
30. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 29, wherein said neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Dementia, a disorder associated with protein misfolding, cognitive decline, Mild Cognitive Impairment (MCI), Parkinson's disease with MCI, Huntington's disease, Lewy body disease, Amyotrophic lateral sclerosis (ALS), Prion disease, Motor neuron disease (MND), Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's Ataxia, multiple sclerosis, Idiopathic Intracrani al Hypertension, Cranial neuropathi es, Trigeminal neuralgia, frontotemporal dementias (FTD), Senile Dementia (Dementia NOS), Motor Neuron Disease, or bipolar disorder.
31. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 30, wherein said neurodegenerative disease is Alzheimer's disease.
32. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 31, for preventing, treating, ameliorating, or delaying the onset of cognitive decline.
33. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 32, wherein said functional fragment or derivative has at least 70%
identity to the amino acid sequence denoted by SEQ ID NO. 1.
identity to the amino acid sequence denoted by SEQ ID NO. 1.
34. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 32, wherein said polypeptide consists of the amino acid sequence denoted by SEQ ID NO. 1.
35. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 34, wherein said polypeptide is able to penetrate the Blood Brain B arri er.
36. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 35, wherein said method results in protection from oxidative stress in the neurons of said subject.
37. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to claim 36, wherein said peptide modulates the expression of at least one gene associated with protection from oxidative stress.
38. The i sol ated polypepti de, functional fragment or derivative or salt thereof for use according to claim 37, wherein said at least one gene is any one of SOD1, BDNF and TH
39. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 38, wherein said method results in increasing glucose metabolism and/or transport in the neurons of said subject.
40. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to claim 39, wherein peptide modulates the expression of at least one gene associated with glucose metaboli sm and/or transp ort.
41. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to claim 40, wherein said at least one gene is any one of GLUT1, GLUT3 and P53.
42. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 41, wherein said method comprises administering a pharmaceutical composition comprising said isolated polypeptide or a functional fragment or derivative of said isolated peptide or a salt thereof.
43. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to any one of claims 26 to 42, wherein said method further comprises administering at least one additional therapeutic agent to said subject.
44. An isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of said isolated peptide or a salt thereof for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated peptide or of a salt thereof.
NO: 1 or a functional fragment or derivative of said isolated peptide or a salt thereof for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof, said method comprises administering to said subject an effective amount of said isolated polypeptide, a functional fragment or derivative of said isolated peptide or of a salt thereof.
45. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to claim 44, wherein said functional fragment or derivative has at least 70%
identity to the amino acid sequence denoted by SEQ ID NO. 1.
identity to the amino acid sequence denoted by SEQ ID NO. 1.
46. The isolated polypeptide, functional fragment or derivative or salt thereof for use according to claim 44, wherein said polypeptide consists of the amino acid sequence denoted by SEQ ID
NO. 1.
NO. 1.
47. A pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent, for use in a method for preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
48. A pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment or derivative of said isolated polypeptide or a salt thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent, for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
49. The pharmaceutical composition for use according to claim 47 or 48, wherein said polypeptide consists of the amino acid sequence denoted by SEQ ID NO. 1.
50. The pharmaceutical composition for use according to claim 47 or claim 49, wherein said neurodegenerative disease is Alzheimer's disease.
51. A kit comprising:
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof; and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for at least one of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof; and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for at least one of preventing, treating, ameliorating or delaying the onset of at least one of a neurodegenerative disease, cognitive decline and any conditions or symptoms associated therewith in a subject in need thereof.
52. A kit comprising:
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof; and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
(a) an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or a functional fragment or derivative of the isolated polypeptide or a salt thereof; and optionally (b) at least one pharmaceutically acceptable excipient, carrier or diluent;
for use in a method for improving at least one of cognitive function, short term memory, long term memory, learning function, glucose metabolism and glucose transport in a subject in need thereof.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202063109484P | 2020-11-04 | 2020-11-04 | |
US63/109,484 | 2020-11-04 | ||
PCT/IL2021/051297 WO2022097136A1 (en) | 2020-11-04 | 2021-11-02 | Polypeptides for use in the treatment and prevention of neurodegenerative diseases |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3196886A1 true CA3196886A1 (en) | 2022-05-12 |
Family
ID=81457689
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3196886A Pending CA3196886A1 (en) | 2020-11-04 | 2021-11-02 | Polypeptides for use in the treatment and prevention of neurodegenerative diseases |
Country Status (7)
Country | Link |
---|---|
US (1) | US20240009277A1 (en) |
EP (1) | EP4232066A4 (en) |
JP (1) | JP2023548609A (en) |
KR (1) | KR20240004214A (en) |
CA (1) | CA3196886A1 (en) |
IL (1) | IL302672A (en) |
WO (1) | WO2022097136A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4413991A1 (en) | 2023-02-08 | 2024-08-14 | Consejo Superior De Investigaciones Científicas | Peptide for cognitive improvement |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014128254A1 (en) * | 2013-02-22 | 2014-08-28 | University Of Eastern Finland (Kuopion Kampus) | Il-33 and treatment of neurodegenerative diseases |
-
2021
- 2021-11-02 WO PCT/IL2021/051297 patent/WO2022097136A1/en active Application Filing
- 2021-11-02 US US18/251,652 patent/US20240009277A1/en active Pending
- 2021-11-02 EP EP21888826.1A patent/EP4232066A4/en active Pending
- 2021-11-02 IL IL302672A patent/IL302672A/en unknown
- 2021-11-02 JP JP2023527708A patent/JP2023548609A/en active Pending
- 2021-11-02 KR KR1020237018702A patent/KR20240004214A/en unknown
- 2021-11-02 CA CA3196886A patent/CA3196886A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
IL302672A (en) | 2023-07-01 |
EP4232066A4 (en) | 2024-10-02 |
WO2022097136A1 (en) | 2022-05-12 |
JP2023548609A (en) | 2023-11-17 |
US20240009277A1 (en) | 2024-01-11 |
KR20240004214A (en) | 2024-01-11 |
EP4232066A1 (en) | 2023-08-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107921085B (en) | Methods and compositions for treating aging-related disorders | |
DE69836740T2 (en) | AMYLOID BETA PROTEIN (GLOBULAR CONSTRUCTION AND ITS USE) | |
US10240156B2 (en) | Modulation of synaptic maintenance | |
US20150359849A1 (en) | Methods of increasing neuronal connectivity and/or treating a neurodegenerative condition | |
WO2018178994A1 (en) | Compositions and methods specifically targeting the apolipoprotein e4 (apoe4) and uses thereof in apoe4 associated conditions | |
Park et al. | Kinetically stabilizing mutations in beta tubulins create isotype-specific brain malformations | |
US20240009277A1 (en) | Polypeptides for use in the treatment and prevention of neurodegenerative diseases | |
US9580469B2 (en) | Peptides for the treatment and early diagnosis of Alzheimer's disease and other tauopathies | |
Niewiadomska et al. | Cytoskeletal transport in the aging brain: focus on the cholinergic system | |
WO2013021196A2 (en) | Oligodendrocyte differentiation | |
US20230399380A1 (en) | Methods and Compositions for Treating Aging-Associated Impairments with TIMP-2 Recombinant Proteins | |
Ager et al. | Stem Cell Therapy for Alzheimer’s Disease | |
JP6168998B2 (en) | Amyloid β protein-specific production inhibitory polypeptide | |
Xia et al. | Inhibition of beta-amyloid precursor protein gene in SK-N-SH cells by piperlonguminine/dihydropiperlonguminine components separated from Chinese herbal medicine Futokadsura stem | |
EP2670421B1 (en) | Use of icam-1 for prevention or treatment of neurological diseases | |
WO2020040106A1 (en) | Aβ-PROTEIN-SPECIFIC PRODUCTION INHIBITOR | |
Brocardo et al. | Altered adult neurogenesis in neurodegenerative diseases | |
Dwyer | Oligodendrocyte dysfunction in dementia and Alzheimer’s disease | |
Obasse | Developing peptide-based inhibitors of amylin aggregation as a novel treatment for type 2 diabetes | |
US20200255492A1 (en) | Leptin peptides and their use for treating neurological disorders | |
JP2021070664A (en) | Brain function improving composition, composition for preventing, treating or improving dementia, and food composition, pharmaceutical composition or cosmetic composition containing them | |
CN118369109A (en) | Peptides and fragments for the treatment of diabetes and related metabolic disorders |